3-aminocyclopentanecarboxamides as modulators of chemokine receptors

ABSTRACT

The present invention is directed to compounds of Formula I: 
                         
which are modulators of chemokine receptors. The compounds of the invention, and compositions thereof, are useful in the treatment of diseases related to chemokine receptor expression and/or activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. Ser. No. 11/167,329, filed Jun.27, 2005, which claims benefit of U.S. Ser. Nos. 60/583,482, filed Jun.28, 2004, and 60/624,481, filed Nov. 1, 2004, the disclosures of whichare incorporated herein by reference in their entireties.

FIELD OF THF INVENTION

The present invention relates to compounds that modulate the activity ofchemokine receptors such as CCR2 and CCR5. In some embodiments, thecompounds modulate both CCR2 and CCR5. The compounds can be used, forexample, to treat diseases associated with chemokine receptor expressionor activity.

BACKGROUND OF THF INVENTION

The migration and transport of leukocytes from blood vessels intodiseased tissues is involved in the initiation of normaldisease-fighting inflammatory responses. The process, also known asleukocyte recruitment, is also related to the onset and progression oflife-threatening inflammatory, as well as debilitating autoimmunediseases. The resulting pathology of these diseases derives from theattack of the body's immune system defenses on normal tissues.Accordingly, preventing and blocking leukocyte recruitment to targettissues in inflammatory, autoimmune disease and cancer would be a highlyeffective approach to therapeutic intervention.

The different classes of leukocyte cells that are involved in cellularimmune responses include monocytes, lymphocytes, neutrophils,eosinophils and basophils. In most cases, lymphocytes are the leukocyteclass that initiates, coordinates, and maintains chronic inflammatoryresponses, and blockage of these cells from entering inflammatory sitesis desirable. Lymphocytes attract monocytes to the tissue sites, which,collectively with lymphocytes, are responsible for most of the actualtissue damage that occurs in inflammatory disease. Infiltration of thelymphocytes and/or monocytes is known to lead to a wide range ofchronic, autoimmune diseases, and also organ transplant rejection. Thesediseases include, but are not limited to, rheumatoid arthritis, chroniccontact dermatitis, inflammatory bowel disease, lupus, systemic lupuserythematosus, multiple sclerosis, atherosclerosis, psoriasis,sarcoidosis, idiopathic pulmonary fibrosis, dermatomyositis, skinpemphigoid and related diseases, (e.g., Pemphigus vulgaris, P.foliacious, P. erythematosis), glomerulonephritides, vasculitides,hepatitis, diabetes, allograft rejection, and graft-versus-host disease.

The process by which leukocytes leave the bloodstream, accumulate atinflammatory sites, and start disease is believed to have at least threesteps which have been described as (1) rolling, (2) activation/firmadhesion and (3) transendothelial migration [Springer, T. A., Nature346:425-433 (1990); Lawrence and Springer, Cell 65:859-873 (1991);Butcher, E. C., Cell 67:1033-1036 (1991)]. The second step is mediatedat the molecular level by chemoattractant receptors. Chemoattractantreceptors on the surface of leukocytes then bind chemoattractantchemokines which are secreted by cells at the site of damage orinfection. Receptor binding activates leukocytes, increases theadhesiveness of the adhesion molecules that mediate transendothelialmigration, and promotes directed migration of the cells toward thesource of the chemoattractant chemokine.

Chemotactic chemokines (leukocyte chemoattractant/activating factors)also known as chemokines, also known as intercrines and SIS chemokines,are a group of inflammatory/immunomodulatory polypeptide factors ofmolecular weight 6-15 kDa that are released by a wide variety of cellssuch as macrophages, monocytes, eosinophils, neutrophils, fibroblasts,vascular endotherial cells, smooth muscle cells, and mast cells, atinflammatory sites (reviewed in Luster, New Eng. J Med., 338, 436-445(1998) and Rollins, Blood, 90, 909-928 (1997)). Also, chemokines havebeen described in Oppenheim, J. J. et al., Annu. Rev. Immunol.,9:617-648 (1991); Schall and Bacon, Curr. Opin. Immunol., 6:865-873(1994); Baggiolini, M., et al., and Adv. Immunol., 55:97-179 (1994).Chemokines have the ability to stimulate directed cell migration, aprocess known as chemotaxis. Each chemokine contains four cysteineresidues (C) and two internal disulfide bonds. Chemokines can be groupedinto two subfamilies, based on whether the two amino terminal cysteineresidues are immediately adjacent (CC family) or separated by one aminoacid (CXC family). These differences correlate with the organization ofthe two subfamilies into separate gene clusters. Within each genecluster, the chemokines typically show sequence similarities between 25to 60%. The CXC chemokines, such as interleukin-8 (IL-8),neutrophil-activating protein-2 (NAP-2) and melanoma growth stimulatoryactivity protein (MGSA) are chemotactic primarily for neutrophils and Tlymphocytes, whereas the CC chemokines, such as RANTES, MIP-1α, MIP-1β,the monocyte chemotactic proteins (MCP-1, MCP-2, MCP-3, MCP-4, andMCP-5) and the eotaxins (−1 and −2) are chemotactic for, among othercell types, macrophages, T lymphocytes, eosinophils, dendritic cells,and basophils. There also exist the chemokines lymphotactin-1,lymphotactin-2 (both C chemokines), and fractalkine (a CXXXC chemokine)that do not fall into either of the major chemokine subfamilies.

MCP-1 (also known as MCAF (abbreviation for macrophage chemotactic andactivating factor) or JE) is a CC chemokine produced bymonocytes/macrophages, smooth muscle cells, fibroblasts, and vascularendothelial cells and causes cell migration and cell adhesion ofmonocytes (see for example Valente, A. J., et al., Biochemistry; 1988,27, 4162; Matsushima, K., et al., J. Exp. Med., 1989, 169, 1485;Yoshimura, T., et al., J. Immunol., 1989, 142, 1956; Rollins, B. J., etal., Proc. Natl. Acad. Sci. USA, 1988, 85, 3738; Rollins, B. J., et al.,Blood, 1991, 78, 1112; Jiang, Y., et al., J. Immunol., 1992, 148, 2423;Vaddi, K., et al., J. Immunol., 1994, 153, 4721), memory T lymphocytes(see for example Carr, M. W., et al., Proc. Natl. Acad. Sci. USA, 1994,91, 3652), T lymphocytes (see for example Loetscher, P., et al., FASEBJ., 1994, 8, 1055) and natural killer cells (see for example Loetscher,P., et al., J. Immunol., 1996, 156, 322; Allavena, P., et al., Eur. J.Immunol., 1994, 24, 3233), as well as mediating histamine release bybasophils (see for example Alam, R., et al., J. Clin. Invest., 1992, 89,723; Bischoff, S. C., et al., J. Exp. Med., 1992, 175, 1271; Kuna, P.,et al., J. Exp. Med., 1992, 175, 489). In addition, high expression ofMCP-1 has been reported in diseases where accumulation ofmonocyte/macrophage and/or T cells is thought to be important in theinitiation or progression of diseases, such as atherosclerosis (see forexample Hayes, I. M., et al., Arterioscler. Thromb. Vasc. Biol., 1998,18, 397; Takeya, M. et al., Hum. Pathol., 1993, 24, 534; Yla-Herttuala,S., et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 5252; Nelken, N. A.,J. Clin. Invest., 1991, 88, 1121), rheumatoid arthritis (see for exampleKoch, A. E., et al., J. Clin. Invest., 1992, 90, 772; Akahoshi, T., etal., Arthritis Rheum., 1993, 36, 762; Robinson, E., et al., Clin. Exp.Immunol., 101, 398), nephritis (see for example Noris, M., et al., Lab.Invest., 1995, 73, 804; Wada, T., at al., Kidney Int., 1996, 49, 761;Gesualdo, L., et al., Kidney Int., 1997, 51, 155), nephropathy (see forexample Saitoh, A., et al., J. Clin. Lab. Anal., 1998, 12, 1; Yokoyama,H., et al., J. Leukoc. Biol., 1998, 63, 493), pulmonary fibrosis,pulmonary sarcoidosis (see for example Sugiyama, Y., et al., InternalMedicine, 1997, 36, 856), asthma (see for example Karina, M., et al., J.Invest. Allergol. Clin. Immunol., 1997, 7, 254; Stephene, T. H., Am. J.Respir. Crit. Care Med., 1997, 156, 1377; Sousa, A. R., et al., Am. J.Respir. Cell Mol. Biol., 1994, 10, 142), multiple sclerosis (see forexample McManus, C., et al., J. Neuroimmunol., 1998, 86, 20), psoriasis(see for example Gillitzer, R., et al., J. Invest. Dermatol., 1993, 101,127), inflammatory bowel disease (see for example Grimm, M. C., et al.,J. Leukoc. Biol., 1996, 59, 804; Reinecker, H. C., et al.,Gastroenterology, 1995, 106, 40), myocarditis (see for example Seino,Y., et al., Chemokine, 1995, 7, 301), endometriosis (see for exampleJolicoeur, C., et al., Am. J. Pathol., 1998, 152, 125), intraperitonealadhesion (see for example Zeyneloglu, H. B., et al., Human Reproduction,1998, 13, 1194), congestive heart failure (see for example Aurust, P.,et al., Circulation, 1998, 97, 1136), chronic liver disease (see forexample Marra, F., et al., Am. J. Pathol., 1998, 152, 423), viralmeningitis (see for example Lahrtz, F., et al., Eur. J. Immunol., 1997,27, 2484), Kawasaki disease (see for example Wong, M.; et al., J.Rheumatol., 1997, 24, 1179) and sepsis (see for example Salkowski, C.A.; et al., Infect. Immun., 1998, 66, 3569). Furthermore, anti-MCP-1antibody has been reported to show an inhibitory effect or a therapeuticeffect in animal models of rheumatoid arthritis (see for exampleSchimmer, R. C., et alt, J. Immunol., 1998, 160, 1466; Schrier, D. J.,J. Leukoc. Biol., 1998, 63, 359; Ogata, H., et al., J. Pathol., 1997,182, 106), multiple sclerosis (see for example Karpus, W. J., et al., J.Leukoc. Biol., 1997, 62, 681), nephritis (see for example Lloyd, C. M.,et al., J. Exp. Med., 1997, 185, 1371; Wada, T., et al., FASEB J., 1996,10, 1418), asthma (see for example Gonzalo, J.-A., et al., J. Exp. Med.,1998, 188, 157; Lukacs, N. W., J. Immunol., 1997, 158, 4398),atherosclerosis (see for example Guzman, L. A., et al., Circulation,1993, 88 (suppl.), I-371), delayed type hypersensitivity (see forexample Rand, M. L., et al., Am. J. Pathol., 1996, 148, 855), pulmonaryhypertension (see for example Kimura, H., et al., Lab. Invest, 1998, 78,571), and intraperitoneal adhesion (see for example Zeyneloglu, H. B.,et al., Am. J. Obstet. Gynecol., 1998, 179, 438). A peptide antagonistof MCP-1, MCP-1(9-76), has been also reported to inhibit arthritis inthe mouse model (see Gong, J.-H., J. Exp., 4 ed., 1997, 186, 131), aswell as studies in MCP-1-deficient mice have shown that MCP-1 isessential for monocyte recruitment in vivo (see Lu, B., et al., J. Exp.Med., 1998, 187, 601; Gu, L., et al., Moll. Cell, 1998, 2, 275).

Chronic obstructive pulmonary disease (COPD) ranks among the most commoncauses of death in Western societies. It is defined by a progressivedecline in lung function, only partly reversible by bronchodilatordrugs. COPD is characterized by chronic inflammation in the airways oralveoli that differs from that seen in asthma, involving increasednumbers of neutrophils, macrophages, CD8+ T cells, and/or mast cells inthe airway walls, alveolar compartments, and vascular smooth muscle.Cytokines associated with COPD are believed to include tumor necrosisfactor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-1 beta,IL-6, IL-8 and MCP-1. CCR2 is known to be a receptor for MCP-1, andrecent data support a role for MCP-1 and CCR2 in airway remodeling andinflammation directly or via macrophages. Thus, antagonists of CCR2 arean attractive approach to therapeutic treatment of COPD (De Boer, W. I,Chest, 2002, 121, 209S-218S).

The literature indicates that chemokines such as MCP-1 and MIP-1αattract monocytes and lymphocytes to disease sites and mediate theiractivation and thus are thought to be intimately involved in theinitiation, progression and maintenance of diseases deeply involvingmonocytes and lymphocytes, such as atherosclerosis, restenosis,rheumatoid arthritis, psoriasis, asthma, ulcerative colitis, nephritis(nephropathy), multiple sclerosis, pulmonary fibrosis, myocarditis,hepatitis, pancreatitis, sarcoidosis, Crohn's disease, endometriosis,congestive heart failure, viral meningitis, cerebral infarction,neuropathy, Kawasaki disease, and sepsis (see for example Rovin, B. H.,et al., Am. J. Kidney. Dis., 1998, 31, 1065; Lloyd, C., et al., Curt.Opin. Nephrol. Hypertens., 1998, 7, 281; Conti, P., et al., Allergy andAsthma Proc., 1998, 19, 121; Ransohoff, R. M., et al., Trends Neurosci.,1998, 21, 154; MacDermott, R. P., et al., Inflammatory Bowel Diseases,1998, 4, 54).

The chemokines bind to specific cell-surface receptors belonging to thefamily of G-protein-coupled seven-transmembrane-domain proteins(reviewed in Horuk, Trends Pharm. Sci., 15, 159-165 (1994)) which aretermed “chemokine receptors.” On binding their cognate ligands,chemokine receptors transduce an intracellular signal through theassociated trimeric G proteins, resulting in, among other responses, arapid increase in intracellular calcium concentration, changes in cellshape, increased expression of cellular adhesion molecules,degranulation, and promotion of cell migration.

Genes encoding receptors of specific chemokines have been cloned, and itis known that these receptors are G protein-coupled seven-transmembranereceptors present on various leukocyte populations. So far, at leastfive CXC chemokine receptors (CXCR1-CXCR5) and eight CC chemokinereceptors (CCR1-CCR10) have been identified. For example IL-8 is aligand for CXCR1 and CXCR2, MIP-1α is a ligand for CCR1 and CCR5, andMCP-1 is a ligand for CCR2A and CCR2B (for reference, see for example,Holmes, W. E., et al., Science 1991, 253, 1278-1280; Murphy P. M., etal., Science, 253, 1280-1283; Neote, K. et al, Cell, 1993, 72, 415-425;Charo, I. F., et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 2752-2756;Yamagami, S., et al., Biochem. Biophys. Res. Commun., 1994, 202,1156-1162; Combadier, C., et al., The Journal of Biological Chemistry,1995, 270, 16491-16494, Power, C. A., et al., J. Biol. Chem., 1995, 270,19495-19500; Samson, M., et al., Biochemistry, 1996, 35, 3362-3367;Murphy, P. M., Annual Review of Immunology, 1994, 12, 592-633). It hasbeen reported that lung inflammation and granuroma formation aresuppressed in CCR1-deficient mice (see Gao, J.-L., et al., J. Exp. Med.,1997, 185, 1959; Gerard, C., et al., J. Clin. Invest., 1997, 100, 2022),and that recruitment of macrophages and formation of atheroscleroticlesion decreased in CCR2-deficient mice (see Boring, L., et al., Nature,1998, 394, 894; Kuziel, W. A., et al., Proc. Natl. Acad. Sci., USA,1997, 94, 12053; Kurihara, T., et al., J. Exp. Med., 1997, 186, 1757;Boring, L., et al., J. Clin. Invest., 1997, 100, 2552).

Chemokine receptors are also known as coreceptors for viral entryleading to viral infection such as, for example, HIV infection. Reversetranscription and protein processing are the classic steps of the virallife cycle which antiretroviral therapeutic agents are designed toblock. Although many new drugs that are believed to block viral entryhold promise, there is currently no agent to which HIV-1 has not beenable to acquire resistance. Multiple rounds of viral replication arerequired to generate the genetic diversity that forms the basis ofresistance. Combination therapy in which replication is maximallysuppressed remains a cornerstone of treatment with entry inhibitors, aswith other agents. The targeting of multiple steps within the viralentry process is believed to have the potential for synergy(Starr-Spires et al., Clin. Lab. Med., 2002, 22(3), 681.)

HIV-1 entry into CD4(+) cells requires the sequential interactions ofthe viral envelope glycoproteins with CD4 and a coreceptor such as thechemokine receptors CCR5 and CXCR4. A plausible approach to blockingthis process is to use small molecule antagonists of coreceptorfunction. The TAK-779 molecule is one such antagonist of CCR5 that actsto prevent HIV-1 infection. TAK-779 inhibits HIV-1 replication at themembrane fusion stage by blocking the interaction of the viral surfaceglycoprotein gp120 with CCR5. The binding site for TAK-779 on CCR5 islocated near the extracellular surface of the receptor, within a cavityformed between transmembrane helices 1, 2, 3, and 7 (Dragic et al.,Proc. Natl. Acad. Sci. USA, 2000, 97(10), 5639).

The chemokine receptors CXCR4 and CCR5 are believed to be used asco-receptors by the T cell-tropic (X4) and macrophage-tropic (R5) HIV-1strains, respectively, for entering their host cells. Propagation of R5strains of HIV-1 on CD4 lymphocytes and macrophages requires expressionof the CCR5 coreceptor on the cell surface. Individuals lacking CCR5(CCR5 Delta 32 homozygous genotype) are phenotypically normal andresistant to infection with HIV-1. Viral entry can be inhibited by thenatural ligands for CXCR4 (the CXC chemokine SDF-1) and CCR5 (the CCchemokines RANTES, MIP-1 alpha and MIP-1beta). The first non-peptidiccompound that interacts with CCR5, and not with CXCR4, is a quaternaryammonium derivative, called TAK-779, which also has potent but variableanti-HIV activity (De Clercq et al., Antivir. Chem. Chemother. 2001, 12Suppl. 1, 19.

SCH-C (SCH 351125) is another small molecule inhibitor of HIV-1 entryvia the CCR5 coreceptor. SCH-C, an oxime-piperidine compound, is aspecific CCR5 antagonist as determined in multiple receptor binding andsignal transduction assays. This compound specifically inhibits HIV-1infection mediated by CCR5 in U-87 astroglioma cells but has no effecton infection of CXCR4-expressing cells. (Strizki et al, Proc. Natl.Acad. Sci. USA, 2001, 98(22), 12718 or Tremblay et al., AntimicrobialAgents and Chemotherapy, 2002, 46(5), 1336).

AD101, chemically related to SCH-C, also inhibits the entry of humanimmunodeficiency virus type 1 (HIV-1) via human CCR5. It has been foundthat AD101 inhibits HIV-1 entry via rhesus macaque CCR5 while SCH-C doesnot. Among the eight residues that differ between the human and macaqueversions of the coreceptor, only one, methionine-198, accounts for theinsensitivity of macaque CCR5 to inhibition by SCH-C. Position 198 is inCCR5 transmembrane (TM) helix 5 and is not located within the previouslydefined binding site for AD101 and SCH-C, which involves residues in TMhelices 1, 2, 3, and 7. Based on studies of amino acid substitutions inCCR5, it has been suggested that the region of CCR5 near residue 198 caninfluence the conformational state of this receptor. (Billick et al.,2004, J. Virol., 78(8), 4134).

The identification of compounds that modulate the activity of chemokinereceptors represents a desirable drug design approach for the neededdevelopment of pharmacological agents for the treatment of diseasesassociated with chemokine receptor activity. The compounds of thepresent invention help fulfill these and other needs.

SUMMARY OF THF INVENTION

The present invention provides compounds of Formula I:

or pharmaceutically acceptable salts or prodrugs thereof, whereinconstituent members are provided herein.

The present invention further provides compositions comprising acompound of Formula I and a pharmaceutically acceptable carrier.

The present invention further provides methods of modulating activity ofa chemokine receptor comprising contacting the chemokine receptor with acompound of Formula I.

The present invention further provides methods of treating a diseaseassociated with expression or activity of a chemokine receptor in apatient comprising administering to the patient a therapeuticallyeffective amount of a compound of Formula I.

The present invention further provides methods of treating HIV infectionin a patient comprising administering to said patient a therapeuticallyeffective amount of a compound of Formula I.

DETAILED DESCRIPTION Compounds

The present invention provides, inter alia, compound of Formula I:

or pharmaceutically acceptable salts or prodrugs thereof, wherein:

a dashed line indicates an optional bond;

W is:

V is N, NO or CR⁵;

X is N, NO or CR²;

Y is N, NO or CR³;

Z is N, NO or CR⁴; wherein no more than one of V, X, Y and Z is NO;

R^(A), R^(A1), R^(B) and R^(B1) are each, independently, H, OH, halo,C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy,C₁₋₆ haloalkoxy, heterocyclyl, carbocyclyl, NR¹⁰R¹², NR¹⁰CO₂R¹¹;NR¹⁰CONR¹⁰R¹², NR¹⁰SO₂NR¹⁰R¹², NR¹⁰—SO₂—R¹¹, CN, CONR¹⁰R¹², CO₂R¹⁰, NO₂,SR¹⁰, SOR¹⁰, SO₂R¹⁰; or SO₂—NR¹⁰R¹²;

R¹ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ hydroxyalkyl, —(C₀₋₆alkyl)-O—(C₁₋₆ alkyl), —(C₀₋₆ alkyl)-S—(C₁₋₆ alkyl), —(C₀₋₆ alkyl)-(C₃₋₇cycloalkyl)-(C₀₋₆ alkyl), OH, OR¹⁰, SR¹⁰, COR¹¹, CO₂R¹⁰, CONR¹⁰R¹²,carbocyclyl, heterocyclyl, CN, NR¹⁰R¹², NR¹⁰SO₂R¹⁰, NR¹⁰COR¹⁰,NR¹⁰CO₂R¹⁰, NR¹⁰CONR¹², CR¹⁰R¹¹CO₂R¹⁰ or CR¹⁰R¹¹OCOR¹⁰;

R², R³, R⁴, R⁵ and R⁶ are each, independently, H, OH, halo, C₁₋₆ alkyl,C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ thioalkoxy, NR¹⁰R¹²,NR¹⁰CO₂R¹¹; NR¹⁰CONR¹⁰R¹², NR¹⁰SO₂NR¹⁰R¹², NR¹⁰—SO₂—R¹¹, heterocyclyl,carbocyclyl, carbocyclyloxy, heterocyclyloxy, CN, NO₂, COR¹¹, CONR¹⁰R¹²,CO₂R¹⁰, NO₂, SR¹⁰, SOR¹⁰, SO₂R¹⁰; or SO₂—NR¹⁰R¹²;

R⁷ is H or C₁₋₆ alkyl optionally substituted by 1-3 substituentsselected from halo, OH, CO₂H, CO₂—(C₁₋₆ alkyl), or C₁₋₃ alkoxy;

R⁸ is C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₆ cycloalkyloxy or OH;

R^(8′) is H;

R⁹ and R^(9′) are each, independently, H, C₁₋₆ alkyl, halo, C₁₋₃ alkoxy,C₁₋₃ haloalkoxy, C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyloxy, OH, CO₂R¹⁰,OCOR¹⁰, wherein said C₁₋₆ alkyl is optionally substituted with one ormore substituents selected from F, C₁₋₃ alkoxy, OH or CO₂R¹⁰;

or R⁹ and R^(9′) together with the carbon atom to which they areattached form a 3-7 membered spirocyclyl group;

R¹⁰ is H, C₁₋₆ alkyl, benzyl, phenyl, or C₃₋₆ cycloalkyl, wherein saidC₁₋₆ alkyl, benzyl, phenyl, or C₃₋₆ cycloalkyl is optionally substitutedwith 1-3 selected from halo, OH, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃alkoxy, C₁₋₃ haloalkoxy, CO₂H, and CO₂—(C₁₋₆ alkyl);

R¹¹ is H, OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, benzyl, phenyl, benzyloxy,phenyloxy, C₃₋₆ cycloalkyl or C₃₋₆ cycloalkyloxy, wherein said C₁₋₆alkyl, C₁₋₆ alkoxy, benzyl, phenyl, benzyloxy, phenyloxy, C₃₋₆cycloalkyl or C₃₋₆ cycloalkyloxy, is optionally substituted with 1-3substituents selected from halo, OH, C₁₋₃ alkyl, C₁₋₃ alkoxy, CO₂H,CO₂—(C₁₋₆ alkyl) and CF₃;

R¹² is H, C₁₋₆ alkyl, benzyl, phenyl, or C₃₋₆ cycloalkyl, wherein saidC₁₋₆ alkyl, benzyl, phenyl, or C₃₋₆ cycloalkyl is optionally substitutedwith 1-3 selected from halo, OH, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃alkoxy, C₁₋₃ haloalkoxy, CO₂H, and CO₂—(C₁₋₆ alkyl); and

p is 0 or 1.

The present invention further provides, inter alia, compounds of FormulaII:

or pharmaceutically acceptable salts or prodrugs thereof, wherein:

a dashed line indicates an optional bond;

W is:

X is N, NO or CR²;

Y is N, NO or CR³;

Z is N, NO or CR⁴; wherein no more than one of X, Y and Z is NO;

R^(A), R^(A1), R^(B) and R^(B1) are each, independently, H, OH, halo,C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy,C₁₋₆ haloalkoxy, heterocyclyl, carbocyclyl, NR¹⁰R¹², NR¹⁰CO₂R¹¹;NR¹⁰OCONR¹⁰R¹², NR¹⁰SO₂NR¹⁰R¹², NR¹⁰—SO₂—R¹¹, CN, CONR¹⁰R¹², CO₂R¹⁰,NO₂, SR¹⁰, SOR¹⁰, SO₂R¹⁰, or SO₂—NR¹⁰R¹²;

R¹ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, (C₀₋₆ alkyl)-O—(C₁₋₆ allyl), (C₀₋₆alkyl)-S—(C₁₋₆ alkyl), (C₀₋₆ alkyl)-(C₃₋₇ cycloalkyl)-(C₀₋₆ alkyl), OH,OR¹⁰, SR¹⁰, COR¹¹, CO₂R¹⁰, CONR¹⁰R¹², carbocyclyl, heterocyclyl, CN,NR¹⁰R¹², NR¹⁰SO₂R¹⁰, NR¹⁰COR¹⁰, NR¹⁰CO₂R¹⁰, NR¹⁰CONR¹², CR¹⁰R¹¹CO₂R¹⁰ orCR¹⁰R¹¹OCOR¹⁰;

R², R³, R⁴, R⁵ and R⁶ are each, independently, H, OH, halo, C₁₋₆ alkyl,C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ thioalkoxy, NR¹⁰R¹²,NR¹⁰CO₂R¹¹; NR¹⁰CONR¹⁰R¹², NR¹⁰SO₂NR¹⁰R¹², NR¹⁰—SO₂—R¹¹, heterocyclyl,carbocyclyl, carbocyclyloxy, heterocyclyloxy, CN, NO₂, COR¹¹, CONR¹⁰R¹²,CO₂R¹⁰, NO₂, SR¹⁰, SOR¹⁰, SO₂R¹⁰; or SO₂—NR¹⁰R¹²;

R⁷ is H or C₁₋₆ alkyl optionally substituted by 1-3 substituentsselected from halo, OH, CO₂H, CO₂—(C₁₋₆ alkyl), or C₁₋₃ alkoxy;

R⁸ is C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₆ cycloalkyloxy or OH;

R^(8′) is H;

R⁹ and R^(9′) are each, independently, H, C₁₋₆ alkyl, halo, C₁₋₃ alkoxy,C₁₋₃ haloalkoxy, C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyloxy, OH, CO₂R¹⁰,OCOR¹⁰, wherein said C₁₋₆ alkyl is optionally substituted with one ormore substituents selected from F, C₁₋₃ alkoxy, OH or CO₂R¹⁰;

or R⁹ and R^(9′) together with the carbon atom to which they areattached form a 3-7 membered spirocyclyl group;

R¹⁰ is H, C₁₋₆ alkyl, benzyl, phenyl, or C₃₋₆ cycloalkyl, wherein saidC₁₋₆ alkyl, benzyl, phenyl, or C₃₋₆ cycloalkyl is optionally substitutedwith 1-3 selected from halo, OH, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃alkoxy, C₁₋₃ haloalkoxy, CO₂H, and CO₂—(C₁₋₆ alkyl);

R¹¹ is H, OH, C₁₋₆ alkyl, C₁₋₆ alkoxy, benzyl, phenyl, benzyloxy,phenyloxy, C₃₋₆ cycloalkyl or C₃₋₆ cycloalkyloxy, wherein said C₁₋₆alkyl, C₁₋₆ alkoxy, benzyl, phenyl, benzyloxy, phenyloxy, C₃₋₆cycloalkyl or C₃₋₆ cycloalkyloxy, is optionally substituted with 1-3substituents selected from halo, OH, C₁₋₃ alkyl, C₁₋₃ alkoxy, CO₂H,CO₂—(C₁₋₆ alkyl) and CF₃;

R¹² is H, C₁₋₆ alkyl, benzyl, phenyl, or C₃₋₆ cycloalkyl, wherein saidC₁₋₆ alkyl, benzyl, phenyl, or C₃₋₆ cycloalkyl is optionally substitutedwith 1-3 selected from halo, OH, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃alkoxy, C₁₋₃ haloalkoxy, CO₂H, and CO₂—(C₁₋₆ alkyl); and

p is 0 or 1.

In some embodiments, W is

In some embodiments, W is

In some embodiments, V is CR⁵.

In some embodiments, X is CR².

In some embodiments, Y is CR³.

In some embodiments, Z is CR⁴.

In some embodiments, X is N.

In some embodiments, Z is N.

In some embodiments, both X and Z are N.

In some embodiments, X is CR²; Y is CR³; and Z is CR⁴.

In some embodiments, V is CR⁵, X is CR²; Y is CR³; and Z is CR⁴.

In some embodiments, no more than 2 of V, X, Y, and Z are N.

In some embodiments, at least 2 of V, X, Y, and Z are other than N orNO.

In some embodiments, none of V, X, Y, and Z are N or NO.

In some embodiments, 1 of V, X, Y, and Z is N.

In some embodiments, 2 of V, X, Y, and Z are N.

In some embodiments, R^(A), R^(A1), R^(B) and R^(B1) are each,independently, H, OH, halo, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, C₁₋₆haloalkyl, C₁₋₆ alkoxy, or C₁₋₆ haloalkoxy.

In some embodiments, R^(A), R^(A1), R^(B) and R^(B1) are each,independently, H, OH or C₁₋₆ alkoxy.

In some embodiments, R^(A), R^(A1), R^(B) and R^(B1) are each,independently, H or OH.

In some embodiments, R¹ is C₁₋₆ alkyl, C₁₋₆ hydroxyalkyl, —(C₀₋₆alkyl)-O—(C₁₋₆ alkyl), or heterocyclyl.

In some embodiments, R¹ is C₁₋₆ alkyl.

In some embodiments, R¹ is prop-2-yl.

In some embodiments, R⁵ and R⁶ is other than H.

In some embodiments, R⁵ and R⁶ is C₁₋₄ haloalkyl.

In some embodiments, R⁶ is C₁₋₄ haloalkyl.

In some embodiments, R⁶ is CF₃.

In some embodiments, R⁷ is H.

In some embodiments, R⁸ is C₁₋₃ alkoxy or C₁₋₃ haloalkoxy.

In some embodiments, R⁸ is C₁₋₃ alkoxy.

In some embodiments, R⁸ is methoxy.

In some embodiments, R⁸ is ethoxy.

In some embodiments, R⁹ and R^(9′) are both H.

In some embodiments, p is 0.

In some embodiments, p is 1.

In some embodiments, compounds of the invention have Formula Ia:

In some embodiments, compounds of the invention have Formula Ib, Ic orId:

In some embodiments, compounds of the invention have Formula Ie, or If:

In some embodiments, compounds of the invention have Formula Ig:

In some embodiments, compounds of the invention have Formula Ih or Ii:

At various places in the present specification, substituents ofcompounds of the invention are disclosed in groups or in ranges. It isspecifically intended that the invention include each and everyindividual subcombination of the members of such groups and ranges. Forexample, the term “C₁₋₆ alkyl” is specifically intended to individuallydisclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl, and C₆ alkyl.

For compounds of the invention in which a variable appears more thanonce, each variable can be a different moiety selected from the Markushgroup defining the variable. For example, where a structure is describedhaving two R groups that are simultaneously present on the samecompound; the two R groups can represent different moieties selectedfrom the Markush group defined for R.

It is further appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, canalso be provided in combination in a single embodiment. Conversely,various features of the invention which are, for brevity, described inthe context of a single embodiment, can also be provided separately orin any suitable subcombination.

As used herein, the term “alkyl” is meant to refer to a saturatedhydrocarbon group which is straight-chained or branched. Example alkylgroups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl andisopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g.,n-pentyl, isopentyl, neopentyl), and the like. An alkyl group cancontain from 1 to about 20, from 2 to about 20, from 1 to about 10, from1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3carbon atoms.

As used herein, “alkylene” refers to a divalent alkyl group.

As used herein, “C₂₋₄ alkylene” refers to an alkylene group comprised offrom 2 to 4 carbon atoms.

As used herein, “alkenyl” refers to an alkyl group having one or moredouble carbon-carbon bonds. Example alkenyl groups include ethenyl,propenyl, cyclohexenyl, and the like.

As used herein, “alkynyl” refers to an alkyl group having one or moretriple carbon-carbon bonds. Example alkynyl groups include ethynyl,propynyl, and the like.

As used herein, “haloalkyl” refers to an alkyl group having one or morehalogen substituents. Example haloalkyl groups include CF₃, C₂F₅, CHF₂,CCl₃, CHCl₂, C₂Cl₅, and the like.

As used herein, “aryl” refers to monocyclic or polycyclic (e.g., having2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example,phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and thelike. In some embodiments, aryl groups have from 6 to about 20 carbonatoms.

As used herein, “carbocyclyl” groups are saturated (i.e., containing nodouble or triple bonds) or unsaturated (i.e., containing one or moredouble or triple bonds) cyclic hydrocarbon moieties. Carbocyclyl groupscan be mono-, poly- (e.g., 2, 3 or 4 fused rings). Example carbocyclylgroups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl,norbornyl, norpinyl, norcarnyl, adamantyl, phenyl, and the like.Carbocyclyl groups can be aromatic (e.g., “aryl”) or non-aromatic (e.g.,“cycloalkyl”). In some embodiments, carbocyclyl groups can have fromabout 3 to about 30 carbon atoms, about 3 to about 20, about 3 to about10, or about 3 to about 7 ring-forming carbon atoms.

As used herein, “cycloalkyl” refers to non-aromatic carbocyclesincluding cyclized alkyl, alkenyl, and alkynyl groups. Cycloalkyl groupscan include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings)ring systems as well as spiro ring systems. Example cycloalkyl groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl,norbornyl, norpinyl, norcarnyl, adamantyl, and the like. Also includedin the definition of cycloalkyl are moieties that have one or morearomatic rings fused (i.e., having a bond in common with) to thecycloalkyl ring, for example, benzo derivatives of pentane, pentene,hexane, and the like. In some embodiments, cycloalkyl groups can havefrom about 3 to about 10, about 3 to about 10, or about 3 to about 7ring-forming carbon atoms. In some embodiments, the cycloalkyl group canhave 0, 1, 2, 3, 4 or 5 double or triple bonds. In yet furtherembodiments, one or more ring-forming carbon atoms of a cycloalkyl groupcan be substituted by an oxo or sulfido group.

As used herein, “heterocyclyl” or “heterocycle” refers to a saturated orunsaturated cyclic hydrocarbon wherein one or more of the ring-formingcarbon atoms of the cyclic hydrocarbon is replaced by a heteroatom suchas O, S, or N. Heterocyclyl groups can be aromatic (e.g., “heteroaryl”)or non-aromatic (e.g., “heterocycloalkyl”). Heterocyclyl groups can alsocorrespond to hydrogenated and partially hydrogenated heteroaryl groups.Heterocyclyl groups can include mono- or polycyclic (e.g., having 2, 3or 4 fused rings) ring systems. Heterocyclyl groups can be characterizedas having 3-14 or 3-7 ring-forming atoms. In some embodiments,heterocyclyl groups can contain, in addition to at least one heteroatom,from about 1 to about 13, about 2 to about 10, or about 2 to about 7carbon atoms and can be attached through a carbon atom or heteroatom. Infurther embodiments, any ring-forming carbon or heteroatom can beoxidized (e.g., have an oxo or sulfido substituent), or a nitrogen atomcan be quaternized. Examples of heterocyclyl groups include morpholino,thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl,2,3-dihydrobenzofuryl, 1,3-benzodioxole, benzo-1,4-dioxane, piperidinyl,pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl,oxazolidinyl, thiazolidinyl, imidazolidinyl, and the like, as well asany of the groups listed below for “heteroaryl” and “heterocycloalkyl.”Further example heterocycles include pyrimidinyl, phenanthridinyl,phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl,phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl,3,6-dihydropyridyl, 1,2,3,6-tetrahydropyridyl,1,2,5,6-tetrahydropyridyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thia-diazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl,xanthenyl, octahydro-isoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxazolidinyl, quinazolinyl, quinolinyl, 4H-quinolizinyl,quinoxalinyl, quinuclidinyl, acridinyl, azocinyl, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzo-thiophenyl, benzoxazolyl,benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,benzisothiazolyl, benzimidazolinyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, deca-hydroquinolinyl, 2H,6H-1,5,2dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, carbazolyl,4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl,indolinyl, indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl and isoxazolyl. Further examples of heterocycles includeazetidin-1-yl, 2,5-dihydro-1H-pyrrol-1-yl, piperindin-1yl,piperazin-1-yl, pyrrolidin-1-yl, isoquinol-2-yl, pyridin-1-yl,3,6-dihydropyridin-1-yl, 2,3-dihydroindol-1-yl,1,3,4,9-tetrahydrocarbolin-2-yl, thieno[2,3-c]pyridin-6-yl,3,4,10,10a-tetrahydro-1H-pyrazino[1,2-a]indol-2-yl,1,2,4,4a,5,6-hexahydro-pyrazino[1,2-a]quinolin-3-yl,pyrazino[1,2-a]quinolin-3-yl, diazepan-1-yl,1,4,5,6-tetrahydro-2H-benzo[f]isoquinolin-3-yl,1,4,4a,5,6,10b-hexahydro-2H-benzo[f]isoquinolin-3-yl,3,3a,8,8a-tetrahydro-1H-2-aza-cyclopenta[a]inden-2-yl, and2,3,4,7-tetrahydro-1H-azepin-1-yl, azepan-1-yl.

As used herein, “heteroaryl” groups refer to an aromatic heterocyclehaving at least one heteroatom ring member such as sulfur, oxygen, ornitrogen. Heteroaryl groups include monocyclic and polycyclic (e.g.,having 2, 3 or 4 fused rings) systems. Examples of heteroaryl groupsinclude without limitation, pyridyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazinyl, furyl (furanyl), quinolyl, isoquinolyl, thienyl,imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl,benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl,tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, benzothienyl,purinyl, carbazolyl, benzimidazolyl, indolinyl, and the like. In someembodiments, the heteroaryl group has from 1 to about 20 carbon atoms,and in further embodiments from about 3 to about 20 carbon atoms. Insome embodiments, the heteroaryl group contains 3 to about 14, 3 toabout 7, or 5 to 6 ring-forming atoms. In some embodiments, theheteroaryl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms.

As used herein, “heterocycloalkyl” refers to non-aromatic heterocyclesincluding cyclized alkyl, alkenyl, and alkynyl groups where one or moreof the ring-forming carbon atoms is replaced by a heteroatom such as anO, N, or S atom. Example “heterocycloalkyl” groups include morpholino,thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl,2,3-dihydrobenzofuryl, 1,3-benzodioxole, benzo-1,4-dioxane, piperidinyl,pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl,oxazolidinyl, thiazolidinyl, imidazolidinyl, and the like. Also includedin the definition of heterocycloalkyl are moieties that have one or morearomatic rings fused (i.e., having a bond in common with) to thenonaromatic heterocyclic ring, for example phthalimidyl, naphthalimidyl,and benzo derivatives of heterocycles such as indolene and isoindolenegroups. In some embodiments, the heterocycloalkyl group has from 1 toabout 20 carbon atoms, and in further embodiments from about 3 to about20 carbon atoms. In some embodiments, the heterocycloalkyl groupcontains 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. Insome embodiments, the heterocycloalkyl group has 1 to about 4, 1 toabout 3, or 1 to 2 heteroatoms. In some embodiments, theheterocycloalkyl group contains 0 to 3 double bonds. In someembodiments, the heterocycloalkyl group contains 0 to 2 double or triplebonds.

As used herein, “spirocyclyl” refers to a 3-14 membered cycloalkyl or3-14 membered heterocycloalkyl group sharing one atom with a furthercycloalkyl or heterocycloalkyl group to which it is attached.

As used herein, “halo” or “halogen” includes fluoro, chloro, bromo, andiodo.

As used herein, “alkoxy” refers to an —O-alkyl group. Example alkoxygroups include methoxy, ethoxy, propoxy (e.g., n-propoxy andisopropoxy), t-butoxy, and the like.

As used herein, “thioalkoxy” refers to an —S-alkyl group.

As used herein, “haloalkoxy” refers to an —O-haloalkyl group. An examplehaloalkoxy group is OCF₃.

As used herein, “carbocyclyloxy” refers to —O-carbocyclyl.

As used herein, “cycloalkyloxy” refers to —O-cycloalkyl.

As used herein, “carbocyclylalkyl” refers to alkyl substituted bycarbocyclyl.

As used herein, “aralkyl” or “arylalkyl” refers to an alkyl groupsubstituted by an aryl group.

As used herein, “cycloalkylalkyl” refers to an alkyl group substitutedby an cycloalkyl group.

As used herein, “heterocyclylalkyl” refers to an alkyl moietysubstituted by a heterocarbocyclyl group. Example heterocyclylalkylgroups include “heteroarylalkyl” (alkyl substituted by heteroaryl) and“heterocycloalkylalkyl” (alkyl substituted by heterocycloalkyl). In someembodiments, heterocyclylalkyl groups have from 3 to 24 carbon atoms inaddition to at least one ring-forming heteroatom.

As used herein “oxo” refers to ═O.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent invention that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins, C═N doublebonds, and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentinvention. Cis and trans geometric isomers of the compounds of thepresent invention are described and may be isolated as a mixture ofisomers or as separated isomeric forms.

Resolution of racemic mixtures of compounds can be carried out by any ofnumerous methods known in the art. An example method includes fractionalrecrystallizaion using a “chiral resolving acid” which is an opticallyactive, salt-forming organic acid. Suitable resolving agents forfractional recrystallization methods are, for example, optically activeacids, such as the D and L forms of tartaric acid, diacetyltartaricacid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid orthe various optically active camphorsulfonic acids such asβ-camphorsulfonic acid. Other resolving agents suitable for fractionalcrystallization methods include stereoisomerically pure forms ofα-methylbenzylamine (e.g., S and R forms, or diastereomerically pureforms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine,cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.

Resolution of racemic mixtures can also be carried out by elution on acolumn packed with an optically active resolving agent (e.g.,dinitrobenzoylphenylglycine). Suitable elution solvent composition canbe determined by one skilled in the art.

Compounds of the invention also include tautomeric forms, such asketo-enol tautomers.

Compounds of the invention can also include all isotopes of atomsoccurring in the intermediates or final compounds. Isotopes includethose atoms having the same atomic number but different mass numbers.For example, isotopes of hydrogen include tritium and deuterium.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The present invention also includes pharmaceutically acceptable salts ofthe compounds described herein. As used herein, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts of the present invention include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Thepharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), eachof which is incorporated herein by reference in its entirety.

The present invention also includes prodrugs of the compounds describedherein. As used herein, “prodrugs” refer to any covalently bondedcarriers which release the active parent drug when administered to amammalian subject. Prodrugs can be prepared by modifying functionalgroups present in the compounds in such a way that the modifications arecleaved, either in routine manipulation or in vivo, to the parentcompounds. Prodrugs include compounds wherein hydroxyl, amino,sulfhydryl, or carboxyl groups are bonded to any group that, whenadministered to a mammalian subject, cleaves to form a free hydroxyl,amino, sulfhydryl, or carboxyl group respectively. Examples of prodrugsinclude, but are not limited to, acetate, formate and benzoatederivatives of alcohol and amine functional groups in the compounds ofthe invention. Preparation and use of prodrugs is discussed in T.Higuchi and V. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 ofthe A.C.S. Symposium Series, and in Bioreversible Carriers in DrugDesign, ed. Edward B. Roche, American Pharmaceutical Association andPergamon Press, 1987, both of which are hereby incorporated by referencein their entirety.

Synthesis

Compounds of the invention, including salts, hydrates, and solvatesthereof, can be prepared using known organic synthesis techniques andcan be synthesized according to any of numerous possible syntheticroutes.

The reactions for preparing compounds of the invention can be carriedout in suitable solvents which can be readily selected by one of skillin the art of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,e.g., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected.

Preparation of Compounds of the Invention can Involve the Protection anddeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups can bereadily determined by one skilled in the art. The chemistry ofprotecting groups can be found, for example, in T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 3rd. Ed., Wiley & Sons,Inc., New York (1999), which is incorporated herein by reference in itsentirety.

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C)infrared spectroscopy, spectrophotometry (e.g., UV-visible), or massspectrometry, or by chromatography such as high performance liquidchromatography (HPLC) or thin layer chromatography.

Exemplary synthetic routes to compounds of the invention are provided inSchemes 1-13 below, where constituent members of the depicted formulaeare defined herein.

3-Aminopentanecarboxylic acids of formula 1-5 can be prepared using theprotocol described in Scheme 1. The commercially available carboxylicacid 1-1 can be converted to an ester such as a methyl ester bytreatment with iodomethane/potassium carbonate in DMF. The resultingester 1-2 can be subjected to an alkylation with a halide such as aniodide (R¹I) using a base such as lithium hexamethyldisilazide (LHMDS)to provide the alkylated product 1-3 as a mixture of cis and transdiastereomers (4:1 ratio). The minor trans diastereomer can be removedby crystallization following hydrolysis of the ester to an acid. Theresulting enantiopure acid 1-4 can be subjected to a hydrogenation usinga catalyst such as Pd—C to afford the saturated carboxylic acid 1-5.

Cyclopentanecarboxylic acids of formula 2-5 can be prepared using theprocedures outlined in Scheme 2. The commercially available3-oxocyclopentanecarboxylic acid 2-1 can be converted to an ester suchas methyl ester. The ketone of the resulting ester 2-2 can be protectedby treatment with trimethyl orthoformate in the presence of an acidiccatalyst such as paratoluenesulfonic acid, Alkylation of the resultingketal 2-3 with an alkyl iodide (R¹I) can be accomplished using a basesuch as LHMDS. Hydrolysis of the alkylated ester 2-4 using a base suchas LiOH, NaOH or KOH provides the carboxylic acids of formula 2-5.

Piperazine derivatives can be prepared using the procedures depicted inScheme 3. Coupling of a piperazine derivative of formula 3-2 with aniodobenzene derivative of formula 3-1 using copper(I) iodide andpotassium phosphate gives rise to the intermediate 3-3. Removal of theBoc group using an acid such as HCl in dioxane or TFA provides thepiperazine derivatives of formula 3-4.

Alternatively, piperazine derivatives (formula 4-3) can be prepared bydisplacement of a 2-chloropyridine or 2-chloropyrimidine derivative offormula 4-1 with a piperazine derivative of formula 4-2.

Alternatively, piperazine derivatives can be prepared using a sequenceas illustrated in Scheme 5. The commercially available3,5-dibromopyridine 5-1 can be converted to 3-bromo-5-iodopyridine 5-2by treatment with isopropylmagnesium bromide and iodine. Coupling of theresulting iodo with a piperazine derivative of formula 3-2 can beaccomplished using copper(I) iodide and potassium phosphate. Followingconversion of the bromo of the resulting intermediate 5-3 to iodo usingisopropylmagnesium bromide and iodine, the iodo can be displaced withtrifluoromethyl by treatment with Me₃SiCF₃/CuI/KF/DMF to afford thetrifluoromethylpyridine derivative of formula 5-5. Removal of the Bocusing an acid such as HCl in dioxane or TFA yields the piperazinederivatives of formula 5-6.

Piperidine or tetrahydropyridine derivatives can be synthesized as shownin Scheme 6. Lithiation of a bromo- or iodobenzene derivative of formula6-1 with an alkyllithium such as n-butyllithium or tert-butyllithiumfollowed by quenching with a ketone derivative of formula 6-2 providesthe tertiary alcohol of formula 6-3. Following dehydration using adehydrating agent such as thionyl chloride/pyridine, the resultingolefin 6-4 can be reduced by hydrogenation using a catalyst such as Pdon carbon. Treatment of 6-3, 6-4 and 6-5 with an acid such as HCl indioxane or TFA provides compounds of formulae 6-6, 6-7 and 6-8.

Alternatively, piperidine or tetrahydropyridine derivatives can besynthesized as illustrated in Scheme 7. A commercially available2-chloropyridine or 2-chloropyrilidine derivative of formula 4-1 can beconverted to 2-bromopyridine derivative of formula 7-1 by treatment withBrSiMe₃. Using similar procedures described in Scheme 6, piperidine andtetrahydropyridine derivatives of formula 7-5 and 7-6 can be obtainedfrom 7-1.

Alternatively, piperidine or tetrahydropyridine derivatives can besynthesized as outlined in Scheme 8.3-Nitro-5-trifluoromethylpyridin-2-ol can be obtained by nitration ofthe commercially available 5-trifluoromethylpyridin-2-ol (8-1).Following conversion of the hydroxy group in 8-2 to choro, the resultingchloro compound 8-3 is subjected to a hydrogenation using a catalystsuch as Pd on carbon to give 3-amino-5-trifluoromethylpyridine 8-4.Diazotiiation of 8-4 using NaNO₂/HBr in the presence of Cu(I)Br provides3-bromo-5-trifluoromethylpyridine 8-5. Following the proceduresdescribed in Scheme 6, 8-5 can be converted to piperidine ortetrahydropyridine derivatives of formulae 8-9 and 8-10.

Tetrahydropyrane derivatives can be obtained as illustrated in Scheme 9(where R^(8a) is, e.g., alkyl). The commercially available4-methoxy-3,6-dihydro-2H-pyran 9-1 can be converted to4,4-dimethoxytetrahydro-2H-pyran-2-ol by treatment withm-chloroperbenzoic acid in methanol. Alkylation of 9-2 with an alkylhalide using NaH gives rise to the trialkoxy intermediate 9-3. Treatmentof 9-3 using an acid such as aqueous HCl affords the ketone products offormula 9-4. The ketone 9-4 can be converted to an amine of formula 9-6by reductive amination with aminodiphenylmethane followed byhydrogenation.

Final compounds of formula I can be assembled using the method describedin Scheme 10. A carboxylic acid of formula 1-5 can be condensed with anamine of formula 10-1 using a standard amide formation agent such as BOPor PyBrop (coupling agent). Following removal of the Boc using an acidsuch as HCl or TFA, the resulting amine 10-3 is subjected to a reductiveamination with a ketone of formula 10-4 using a reducing agent such assodium triacetoxyborohydride to provide final compounds of formula 10-5.

Alternatively, compounds of the invention can be assembled according toScheme 11. Coupling of a carboxylic acid of formula 2-5 with an amine offormula 10-1 using a standard amide formation method produces the amideof formula 11-1. Following conversion of the ketal to a ketone using anaqueous acid, reductive amination of the resulting ketone 11-2 with anamine of formula 11-3 using a reducing agent such as sodiumtriacertoxyborohydride provides compounds of formula 11-4.

Methods

In some embodiments, compounds of the invention can modulate activity ofone or more chemokine receptors. The term “modulate” is meant to referto an ability to increase or decrease, activity of a receptor.Accordingly, compounds of the invention can be used in methods ofmodulating a chemokine receptor by contacting the receptor with any oneor more of the compounds or compositions described herein. In someembodiments, compounds of the present invention can act as inhibitors ofchemokine receptors. In further embodiments, the compounds of theinvention can be used to modulate activity of a chemokine receptor in anindividual in need of modulation of the receptor by administering amodulating amount of a compound of Formula I.

Chemokine receptors to which the present compounds bind and/or modulateinclude any chemokine receptor. In some embodiments, the chemokinereceptor belongs to the CC family of chemokine receptors including, forexample, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, and CCR10. Insome embodiments, the chemokine receptor is CCR2. In some embodiments,the chemokine receptor is CCR5. In some embodiments, the chemokinereceptor binds and/or modulates both CCR2 and CCR5.

The compounds of the invention can be selective. By “selective” is meantthat a compound binds to or inhibits a chemokine receptor with greateraffinity or potency, respectively, compared to at least one otherchemokine receptor.

Compounds of the invention can be dual inhibitors or binders of CCR2 andCCR5, meaning that the compounds of the invention can bind to or inhibitboth CCR2 and CCR5 with greater affinity or potency, respectively, thanfor other chemokine receptors such as CCR1, CCR3, CCR4, CCR6, CCR7,CCR8, and CCR10. In some embodiments, the compounds of the inventionhave binding or inhibition selectivity for CCR2 and CCR5 over any otherchemokine receptor. Selectivity can be at least about 10-fold, at leastabout 20-fold, at least about 50-fold, at least about 100-fold, at leastabout 200-fold, at least about 500-fold or at least about 1000-fold.Binding affinity and inhibitor potency can be measured according toroutine methods in the art, such as according to the assays providedherein.

The present invention further provides methods of treating a chemokinereceptor-associated disease or disorder in an individual (e.g., patient)by administering to the individual in need of such treatment atherapeutically effective amount or dose of a compound of the presentinvention or a pharmaceutical composition thereof. A chemokinereceptor-associated disease can include any disease, disorder orcondition that is directly or indirectly linked to expression oractivity of the chemokine receptor. A chemokine receptor-associateddisease can also include any disease, disorder or condition that can beprevented, ameliorated, or cured by modulating chemokine receptoractivity. A chemokine receptor-associated disease can further includeany disease, disorder or condition that is characterized by binding ofan infectious agent such as a virus or viral protein with a chemokinereceptor. In some embodiments, the chemokine receptor-associated diseaseis a CCR5-associated disease such as HIV infection.

Example chemokine receptor-associated diseases, disorders and conditionsinclude inflammation and inflammatory diseases, immune disorders,cancer, and viral infections. Example inflammatory diseases includediseases having an inflammatory component such as asthma, seasonal andperennial allergic rhinitis, sinusitis, conjunctivitis, age-relatedmacular degeneration, food allergy, scombroid poisoning, psoriasis,urticaria, pruritus, eczema, inflammatory bowel disease, thromboticdisease, otitis media, liver cirrhosis, cardiac disease, Alzheimer'sdisease, sepsis, restenosis, atherosclerosis, multiple sclerosis,Crohn's disease, ulcerative colitis, hypersensitivity lung diseases,drug-induced pulmonary fibrosis, chronic obstructive pulmonary disease(COPD), rheumatoid arthritis, and nephritis, ulcerative colitis, atopicdermatitis, stroke, acute nerve injury, sarcoidosis, hepatitis,endometriosis, neuropathic pain, hypersensitivity pneumonitis,eosinophilic pneumonias, delayed-type hypersensitivity, interstitiallung disease (ILD) (e.g., idiopathic pulmonary fibrosis, or ILDassociated with rheumatoid arthritis, systemic lupus erythematosus,ankylosing spondylitis, systemic sclerosis, Sjogren's syndrome,polymyositis or dermatomyositis), eye disorders (e.g., retinalneurodegeneration, choroidal neovascularization, etc.) and the like.Example immune disorders include rheumatoid arthritis, psoriaticarthritis, systemic lupus erythematosus, myastenia gravis, juvenileonset diabetes; glomerulonephritis, autoimmune throiditis, organtransplant rejection including allograft rejection and graft-versus-hostdisease. Example cancers include cancers such as breast cancer, ovariancancer, multiple myeloma and the like that are characterized byinfiltration of macrophages (e.g., tumor associated macrophages, TAMs)into tumors or diseased tissues. Example viral infections include Herpesinfection, HIV infection or AIDS.

As used herein, the term “contacting” refers to the bringing together ofindicated moieties in an in vitro system or an in vivo system. Forexample, “contacting” the chemokine receptor with a compound of theinvention includes the administration of a compound of the presentinvention to an individual or patient, such as a human, having achemokine receptor, as well as, for example, introducing a compound ofthe invention into a sample containing a cellular or purifiedpreparation containing the chemokine receptor.

As used herein, the term “individual” or “patient,” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep,horses, or primates, and most preferably humans.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician, which includes one or more of thefollowing:

(1) preventing the disease; for example, preventing a disease, conditionor disorder in an individual who may be predisposed to the disease,condition or disorder but does not yet experience or display thepathology or symptomatology of the disease (non-limiting examples arepreventing hypersensitivity lung diseases, drug-induced pulmonaryfibrosis, chronic obstructive pulmonary disease (COPD),graft-versus-host disease and/or allograft rejection aftertransplantation, or preventing allergic reactions such as atopicdermatitis, or seasonal or perennial allergic rhinitis);

(2) inhibiting the disease; for example, inhibiting a disease, conditionor disorder in an individual who is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder (i.e.,arresting further development of the pathology and/or symptomatology)such as inhibiting the autoimmune response in hypersensitivity lungdiseases, drug-induced pulmonary fibrosis, chronic obstructive pulmonarydisease (COPD), rheumatoid arthritis, lupus or psoriasis, or inhibitingtumor growth or stabilizing viral load in the case of a viral infection;and

(3) ameliorating the disease; for example, ameliorating a disease,condition or disorder in an individual who is experiencing or displayingthe pathology or symptomatology of the disease, condition or disorder(i.e., reversing the pathology and/or symptomatology) such as decreasingthe autoimmune response in hypersensitivity lung diseases, drug-inducedpulmonary fibrosis, chronic obstructive pulmonary disease (COPD),rheumatoid arthritis, lupus or psoriasis, or shrinking a tumorassociated with cancer or lowering viral load in the case of a viralinfection.

One or more additional pharmaceutical agents such as, for example,antibodies, anti-inflammatory agents, immunosuppressants,chemotherapeutics can be used in combination with the compounds of thepresent invention for treatment of chemokine receptor-associateddiseases, disorders or conditions. The agents can be combined with thepresent compounds in a single dosage form, or the agents can beadministered simultaneously or sequentially as separate dosage forms.

One or more additional pharmaceutical agents such as, for example,anti-viral agents, antibodies, anti-inflammatory agents, insulinsecretagogues and sensitizers, serum lipid and lipid-carrier modulatingagents, and/or immunosuppressants can be used in combination with thecompounds of the present invention for treatment of chemokinereceptor-associated diseases, disorders or conditions. The agents can becombined with the present compounds in a single or continuous dosageform, or the agents can be administered simultaneously or sequentiallyas separate dosage forms.

Suitable antiviral agents contemplated for use in combination with thecompounds of the present invention can comprise nucleoside andnucleotide reverse transcriptase inhibitors (NRTIs), non-nucleosidereverse transcriptase inhibitors (NNRTIs), protease inhibitors and otherantiviral drugs.

Suitable antiviral agents contemplated for use in combination with thecompounds of the present invention can comprise nucleoside andnucleotide reverse transcriptase inhibitors (NRTIs), non-nucleosidereverse transcriptase inhibitors (NNRTIs), protease inhibitors, entryinhibitors, fusion inhibitors, maturation inhibitors, and otherantiviral drugs.

Example suitable NRTIs include zidovudine (AZT); didanosine (ddI);zaleitabine (ddC); stavudine (d4T); lamivudine (3TC); abacavir(1592U89); adefovir dipivoxil [bis(POM)-PMEA]; lobucavir (BMS-180194);BCH-10652; emitricitabine [(−)-FTC]; beta-L-FD4 (also called beta-L-D4Cand named beta-L-2′,3′-dicleoxy-5-fluoro-cytidene); DAPD,((−)-beta-D-2,6,-diamino-purine dioxolane); and lodenosine (FddA).

Typical suitable NNRTIs include nevirapine (BI-RG-587); delaviradine(BHAP, U-90152); efavirenz (DMP-266); PNU-142721; AG-1549; MKC-442(1-(ethoxy-methyl)-5-(1-methylethyl)-6-(phenylmethyl)-(2,4(1H,3H)-pyrimidinedione); and (+)-calanolide A (NSC-675451) and B.

Typical suitable protease inhibitors include saquinavir (Ro 31-8959);ritonavir (ABT-538); indinavir (MK-639); nelfnavir (AG-1343); amprenavir(141W94); lasinavir (BMS-234475); DMP-450; BMS-2322623; ABT-378; andAG-1 549.

Other antiviral agents include hydroxyurea, ribavirin, IL-2, IL-12,pentafuside, enfuvirtide, C-34, the cyclotriazadisulfonamide CADA,PA-457 and Yissum Project No. 11607.

In some embodiments, anti-inflammatory or analgesic agents contemplatedfor use in combination with the compounds of the present invention cancomprise, for example, an opiate agonist, a lipoxygenase inhibitor suchas an inhibitor of 5-lipoxygenase, a cyclooxygenase inhibitor such as acyclooxygenase-2 inhibitor, an interleukin inhibitor such as aninterleukin-1 inhibitor, a TNF inhibitor such as infliximab, etanercept,or adalimumab an NNMA antagonist, an inhibitor of nitric oxide or aninhibitor of the synthesis of nitric oxide, a non-steroidalantiinflammatory agent, or a cytokine-suppressing antiinflammatoryagent, for example, such as acetaminophen, aspirin, codeine, fentanyl,ibuprofen, indomethacin, ketodolac, morphine, naproxen, phenacetin,piroxicam, a steroidal analgesic, sufentanyl, sunlindac, tenidap, andthe like. Similarly, the instant compounds can be administered with apain reliever; a potentiator such as caffeine, an H2-antagonist,simethicone, aluminum or magnesium hydroxide; a decongestant such asphenylephrine, phenylpropanolamine, pseudophedrine, oxymetazoline,ephinephrine, naphazoline, xylometazoline, propylhexedfine, orlevo-desoxyephedrine; an antfitussive such as codeine, hydrocodone,caramiphen, carbetapentane, or dextramethorphan; a diuretic; and asedating or non-sedating antihistamine.

In some embodiments, pharmaceutical agents contemplated for use incombination with the compounds of the present invention can comprise butare not limited to (a) VLA-4 antagonists such as those described in U.S.Pat. No. 5,510,332, W095/15973, W096/01644, W096/06108, W096/20216,W096/229661, W096/31206, W096/4078, W097/030941, W097/022897 WO98/426567 W098/53814, W098/53817, W098/538185, W098/54207, andW098/58902; (b) steroids such as beclornethasone, methylpi-ednisolone,betarnethasone, prednisone, dexamethasone, and hydrocortisone; (c)immunosuppressants such as cyclosporin, tacrolimus, raparnycin and otherFK506 type immunosuppressants; (d) antihistamines (HI-histamineantagonists) such as bromopheniramine, chlorpheniramine,dexchlorpheniramine, triprolidine, clemastine, diphenhydramine,diphenylpyraline, tripelennamine, hydroxyzine, methdilazine,promethazine, trimeprazine, azatadine, cyproheptadine, antazoline,pheniramine pyrilamine, asternizole, terfenadine, loratadine,cetirizine, fexofenadine, desearboethoxyloratadine, and the like; (e)non-steroidal anti-asthmatics such as terbutaline, metaproterenol,fenoterol, isoethaiine, albuterol, bitolterol, pirbuterol, theophylline,cromolyn sodium, atropine, ipratropium bromide, leukotriene antagonists(e.g., zafirlukast, montelukast, pranlukast, iralukast, pobilukast,SKB-106,203), leukotriene biosynthesis inhibitors (e.g., zileuton,BAY-1005); (f) nonsteroidal antiinflammatory agents (NSAIDs) such aspropionic acid derivatives (e.g., alminoprofen, benoxaprofen, bucloxicacid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen,ibuprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin,pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen),acetic acid derivatives (e.g., indomethacin, acernetacin, alclofenac,clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac,ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin,and zomepirac), fenarnic acid derivatives (flufenarnic acid,meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid),biphenylearboxylic acid derivatives (diflunisal and flufenisal),oxicarns (isoxicarn, piroxicam, sudoxicam and tenoxican), salicylates(acetyl salicylic acid, sulfasalazine) and the pyrazolones (apazone,bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone);(g) cyclooxygenase-2 (COX-2) inhibitors; (h) inhibitors ofphosphodiesterase type IV (PDE-IV); (i) other antagonists of thechemokine receptors, especially CXCR-4, CCR1, CCR2, CCR3 and CCR5; (j)cholesterol lowering agents such as HMG-CoA reductase inhibitors(lovastatin, sirrivastatin and pravastatin, fluvastatin, atorvastatin,and other statins), sequestrants (cholestyramine and colestipol),nicotinic acid, fenofibric acid derivatives (gemfibrozil, clofibrat,fenofibrate and benzafibrate), and probucol; (k) anti-inflammatorybiologic agents such as anti-TNF therapies, anti-IL-1 receptor,CTLA-4Ig, anti-CD20, and anti-VLA4 antibodies; (l) anti-diabetic agentssuch as insulin, sulfonylureas, biguanides (metformin), U.-glucosidaseinhibitors (acarbose) and orlitazones (troglitazone and pioglitazone);(m) preparations of interferon beta (interferon beta-1o., interferonbeta-1 P); (n) other compounds such as aminosalicylic acids,antimetabolites such as azathioprine and 6-mercaptopurine, and cytotoxiccancer chemotherapeutic agents. The weight ratio of the compound of thecompound of the present invention to the second active ingredient may bevaried and will depend upon the effective dose of each ingredient.

For example, a CCR2 and/or CCR5 antagonist can be used in combinationwith an anti-inflammatory pharmaceutical agent in the treatment ofinflammation, metabolic disease, autoimmune disease, cancer or viralinfection to improve the treatment response as compared to the responseto the therapeutic agent alone, without exacerbation of its toxiceffects. Additive or synergistic effects are desirable outcomes ofcombining a CCR2 and/or CCR5 antagonist of the present invention with anadditional agent. Furthermore, resistance of cancer cells to agents suchas dexamethasone can be reversible upon treatment with a CCR2 and/orCCR5 antagonist of the present invention.

Pharmaceutical Formulations and Dosage Forms

When employed as pharmaceuticals, the compounds of Formula I can beadministered in the form of pharmaceutical compositions. Thesecompositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routesdepending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration can be topical (includingophthalmic and to mucous membranes including intranasal, vaginal andrectal delivery), pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer, intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal intramuscular or injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Parenteraladministration can be in the form of a single bolus dose, or can be, forexample, by a continuous perfusion pump. Pharmaceutical compositions andformulations for topical administration can include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the compounds of Formula Iabove in combination with one or more pharmaceutically acceptablecarriers. In making the compositions of the invention, the activeingredient is typically mixed with an excipient, diluted by an excipientor enclosed within such a carrier in the form of, for example, acapsule, sachet, paper, or other container. When the excipient serves asa diluent, it can be a solid, semi-solid, or liquid material, which actsas a vehicle, carrier or medium for the active ingredient. Thus, thecompositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments containing, forexample, up to 10% by weight of the active compound, soft and hardgelatin capsules, suppositories, sterile injectable solutions, andsterile packaged powders.

In preparing a formulation, the active compound can be milled to providethe appropriate particle size prior to combining with the otheringredients. If the active compound is substantially insoluble, it canbe milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 1000 mg (1 g), more usually about 100to about 500 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

In some embodiments, the compounds or compositions of the inventioncontain from about 5 to about 50 mg of the active ingredient. One havingordinary skill in the art will appreciate that this embodies compoundsor compositions containing from about 5 to about 10, from about 10 toabout 15, from about 15 to about 20, from about 20 to about 25, fromabout 25 to about 30, from about 30 to about 35, from about 35 to about40, from about 40 to about 45, or from about 45 to about 50 mg of theactive ingredient.

In some embodiments, the compounds or compositions of the inventioncontain from about 50 to about 500 mg of the active ingredient. Onehaving ordinary skill in the art will appreciate that this embodiescompounds or compositions containing from about 50 to about 75, fromabout 75 to about 100, from about 100 to about 125, from about 125 toabout 150, from about 150 to about 175, from about 175 to about 200,from about 200 to about 225, from about 225 to about 250, from about 250to about 275, from about 275 to about 300, from about 300 to about 325,from about 325 to about 350, from about 350 to about 375, from about 375to about 400, from about 400 to about 425, from about 425 to about 450,from about 450 to about 475, or from about 475 to about 500 mg of theactive ingredient.

In some embodiments, the compounds or compositions of the inventioncontain from about 500 to about 1000 mg of the active ingredient. Onehaving ordinary skill in the art will appreciate that this embodiescompounds or compositions containing from about 500 to about 550, fromabout 550 to about 600, from about 600 to about 650, from about 650 toabout 700, from about 700 to about 750, from about 750 to about 800,from about 800 to about 850, from about 850 to about 900, from about 900to about 950, or from about 950 to about 1000 mg of the activeingredient.

The active compound can be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 1000 mg of the activeingredient of the active ingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in can be nebulized by use of inert gases. Nebulizedsolutions may be breathed directly from the nebulizing device or thenebulizing device can be attached to a face masks tent, or intermittentpositive pressure breathing machine. Solution, suspension, or powdercompositions can be administered orally or nasally from devices whichdeliver the formulation in an appropriate manner.

The amount of compound or composition administered to a patient willvary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of the compounds of the present invention canvary according to, for example, the particular use for which thetreatment is made, the manner of administration of the compound, thehealth and condition of the patient, and the judgment of the prescribingphysician. The proportion or concentration of a compound of theinvention in a pharmaceutical composition can vary depending upon anumber of factors including dosage, chemical characteristics (e.g.,hydrophobicity), and the route of administration. For example, thecompounds of the invention can be provided in an aqueous physiologicalbuffer solution containing about 0.1 to about 10% w/v of the compoundfor parenteral administration. Some typical dose ranges are from about 1μg/kg to about 1 g/kg of body weight per day. In some embodiments, thedose range is from about 0.01 mg/kg to about 100 mg/kg of body weightper day. The dosage is likely to depend on such variables as the typeand extent of progression of the disease or disorder, the overall healthstatus of the particular patient, the relative biological efficacy ofthe compound selected, formulation of the excipient, and its route ofadministration. Effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

The compounds of the invention can also be formulated in combinationwith one or more additional active ingredients which can include anypharmaceutical agent such as antibodies, immune suppressants,anti-inflammatory agents, chemotherapeutics, lipid lowering agents, HDLelevating agents, insulin secretagogues or sensitizers, drugs used forthe treatment of rheumatoid arthritis and the like.

Rheumatoid Arthritis (RA) Treatment Regimen

Rheumatoid arthritis (RA) patients, treated aggressively with diseasemodifying agents (methotrexate, antimalarials, gold, penicillamine,sulfasalazine, dapsone, leflunamide, or biologicals), can achievevarying degrees of disease control, including complete remissions. Theseclinical responses are associated with improvement in standardizedscores of disease activity, specifically the ACR criteria whichincludes: pain, function, number of tender joints, number of swollenjoints, patient global assessment, physician global assessment,laboratory measures of inflammation (CRP and ESR), and radiologicassessment of joint structural damage. Current disease-modifying drugs(DMARDs) require continued administration to maintain optimal benefit.Chronic dosing of these agents is associated with significant toxicityand host defense compromise. Additionally, patients often becomerefractory to a particular therapy and require an alternative regimen.For these reasons, a novel, effective therapy which allows withdrawal ofstandard DMARDs would be a clinically important advance.

Patients with significant response to anti-TNF therapies (infliximab,etanercept, adalimumab), anti-IL-1 therapy (kinaret) or other diseasemodifying anti-rheumatic drugs (DMARDs) including but not limited tomethotrexate, cyclosporine, gold salts, antimalarials, penicillamine orleflunamide, who have achieved clinical remission of disease can betreated with a substance that inhibits expression and/or activity ofCCR2 including, for example, nucleic acids (e.g., antisense or siRNAmolecules), proteins (e.g., anti-CCR2 antibodies), small moleculeinhibitors (e.g., the compounds disclosed herein and other chemokinereceptor inhibitors known in the art).

In some embodiments, the substance that inhibits expression and/oractivity of CCR2 is a small molecule CCR2 inhibitor (or antagonist). TheCCR2 antagonist can be dosed orally q.d. or b.i.d at a dose not toexceed about 500 mgs a day. The patients can be withdrawn from or have adecrease in the dosage of their current therapy and would be maintainedon treatment with the CCR2 antagonist. Treating patients with acombination of CCR2 antagonist and their current therapy can be carriedout for, for example, about one to about two days, before discontinuingor dose reducing the DMARD and continuing on CCR2 antagonist.

Advantages of substituting traditional DMARDS with CCR2 antagonists arenumerous. Traditional DMARDs have serious cumulative dose-limiting sideeffects, the most common being damage to the liver, as well asimmunosuppressive actions. CCR2 antagonism is expected to have animproved long-term safety profile and will not have similarimmunosuppressive liabilities associated with traditional DMARDs.Additionally, the half-life of the biologicals is typically days orweeks, which is an issue when dealing with adverse reactions. Thehalf-life of an orally bioavailable CCR2 antagonist is expected to be onthe order of hours so the risk of continued exposure to the drug afteran adverse event is very minimal as compared to biological agents. Also,the current biologic agents (infliximab, etanercept, adalimumab,kinaret) are typically given either i.v. or s.c., requiring doctor'sadministration or patient self-injection. This leads to the possibilityof infusion reaction or injection site reactions. These are avoidableusing an orally administered CCR2 antagonist.

Diabetes and Insulin Resistance Treatment Regimen

Type 2 diabetes is one of the leading causes of morbidity and mortalityin western societies. In the vast majority of patients, the disease ischaracterized by pancreatic beta-cell dysfunction accompanied by insulinresistance in the liver and in peripheral tissues. Based on the primarymechanisms that are associated with disease, two general classes of oraltherapies are available to treat type 2 diabetes: insulin secretagogues(sulfonylureas such as glyburide) and insulin sensitizers (metformin andthiazolidinediones such as rosiglitazone). Combination therapy thataddresses both mechanisms has been shown to manage the metabolic defectsof this disease and in many instances can be shown to ameliorate theneed for exogenous insulin administration. However, with time, insulinresistance often progresses, leading to the need for further insulinsupplementation. In addition, a prediabetic state, referred to as themetabolic syndrome, has been demonstrated to be characterized byimpaired glucose tolerance, particularly in association with obesity.The majority of patients who develop type 2 diabetes begin by developinginsulin resistance, with the hyperglycemia occurring when these patientscan no longer sustain the degree of hyperinsulinemia necessary toprevent loss of glucose homeostasis. The onset of the insulin resistancecomponent is highly predictive of disease onset and is associated withan increase in the risk of developing type 2 diabetes, hypertension andcoronary heart disease.

One of the strongest correlates of impaired glucose tolerance and of theprogression from an insulin resistant state to type 2 diabetes is thepresence of central obesity. Most patients with type 2 diabetes areobese and obesity itself is associated with insulin resistance. It isclear that central adiposity is a major risk factor for the developmentof insulin resistance leading to type 2 diabetes, suggesting thatsignals from visceral fat contribute to the development of insulinresistant and progression to disease. In addition to the secretedprotein factors, obesity induces a cellular inflammatory response inwhich bone-marrow derived macrophages accumulate in adipose depots,becoming adipose tissue macrophages. Adipose tissue macrophagesaccumulate in adipose tissue in proportion to measures of adiposity.Tissue infiltrating macrophages are a source of many of the inflammatorycytokines that have been demonstrated to induce insulin resistance inadipocytes.

Adipose tissue produces MCP-1 in proportion to adiposity, suggestingthat its activity by signaling through CCR2 also might play an importantrole in the accumulation of macrophages in adipose tissue. It is unknownwhether the MCP-1/CCR2 interaction is directly responsible for monocyterecruitment to adipose tissue, whether reduced recruitment ofmacrophages to adipose tissue in humans will directly lead to thereduced production of proinflammatory molecules and whether theproinflammatory molecule production is directly linked to insulinresistance.

Patients who demonstrate insulin resistance, either prediabetic(normoglycemic) or diabetic (hyperglycemic), could be treated with asubstance that inhibits the expression and/or activity of CCR2including, for example, nucleic acids (e.g., antisense or siRNAmolecules), proteins (e.g., anti-CCR2 antibodies), small moleculeinhibitors (e.g., the compounds disclosed herein and other chemokinereceptor inhibitors known in the art). In some embodiments, thesubstance that inhibits expression and/or activity of CCR2 is a smallmolecule CCR2 inhibitor (or antagonist). The CCR2 antagonist can bedosed orally q.d. or bid at a dose not to exceed about 500 mgs a day.The patients can be withdrawn from or have a decrease in the dosage oftheir current therapy and would be maintained on treatment with the CCR2antagonist. Alternately CCR2 antagonist treatment may be used tosupplement their current therapy to enhance its effectiveness or toprevent progression to further insulin dependence.

Advantages of substituting or supplementing traditional agents with CCR2antagonists are numerous. Such agents may be useful, for example, topreclude progression from a prediabetic, insulin resistant state to adiabetic state. Such agents may reduce or replace the need for the useof insulin sensitizers, with their attendant toxicities. Such agents mayalso reduce the need for, or prolong the period until, exogenous insulinsupplementation is required.

Atherosclerosis Treatment Regimen

Atherosclerosis is a condition characterized by the deposition of fattysubstances in arterial walls. Plaque encompasses such deposits of fattysubstances, cholesterol, cellular waste products, calcium and othersubstances that build up in the inner lining of an artery. Plaques cangrow large enough to significantly reduce the blood's flow through anartery. However, more significant damage occurs when the plaque becomesunstable and ruptures. Plaques that rupture cause blood clots to formthat can block blood flow or break off and travel to other parts of thebody. If the clot blocks a blood vessel that feeds the heart, it causesa heart attack. If it blocks a blood vessel that feeds the brain, itcauses a stroke. Atherosclerosis is a slow, complex disease thattypically starts in childhood and often progresses as people grow older.

A high level of cholesterol in the blood is a major risk factor forcoronary heart disease. Based on cholesterol as a primary composition ofplaque, the advance of plaque formation has been managed by thereduction of circulating cholesterol or by elevation ofcholesterol-carrying high density lipoproteins (HDL). Circulatingcholesterol can be reduced, for example, by inhibiting its synthesis inthe liver using or by reducing update from food. Such medicaments thatact through these mechanism may include medicines that are used to lowerhigh cholesterol levels: bile acid absorbers, lipoprotein synthesisinhibitors, cholesterol synthesis inhibitors and fibric acidderivatives. Circulating HDL can additionally be elevated byadministration of, for example, probuchol or high doses of niacin.Therapy that addresses multiple mechanisms has been shown to slowdisease progression and progression to plaque rupture.

Atherosclerosis is typically accompanied by a cellular inflammatoryresponse in which bone-marrow derived macrophages accumulate in fattystreaks along the vessel wall, becoming foam cells. Foam cells are asource of many of the inflammatory cytokines that have been demonstratedto induce plaque progression and of the enzymes that can promote plaquedestabilization. Atherosclerotic tissue also produces MCP-1, suggestingthat its activity by signaling through CCR2 also might play an importantrole in the accumulation of macrophages as foam cells in plaques.CCR2−/− mice have been demonstrated to have significantly reducedmacrophages in fatty streaks generated as a result of high fat diet orgenetic alteration in lipid metabolism.

Patients who demonstrate high circulating cholesterol, low HDL, orelevated circulating CRP or present with vessel wall plaque by imaging,or any other evidence of the presence of atherosclerosis could betreated with a substance that inhibits the expression and/or activity ofCCR2 including, for example, nucleic acids (e.g., antisense or siRNAmolecules), proteins (e.g., anti-CCR2 antibodies), small moleculeinhibitors (e.g., the compounds disclosed herein and other chemokinereceptor inhibitors known in the art). In some embodiments, thesubstance that inhibits expression and/or activity of CCR2 is a smallmolecule CCR2 inhibitor (or antagonist) such as a compound of theinvention. The CCR2 antagonist can be dosed orally q.d. or b.i.d at adose not to exceed about 500 mgs a day. The patients can be withdrawnfrom or have a decrease in the dosage of their current therapy and wouldbe maintained on treatment with the CCR2 antagonist. Alternately CCR2antagonist treatment may be used to supplement their current therapy toenhance its effectiveness in, for example, preventing plaqueprogression, stabilizing plaque that has already formed or inducingplaque regression.

Advantages of substituting or supplementing traditional agents with CCR2antagonists are numerous. Such agents may be useful, for example, topreclude progression of the plaque to a stage of instability with itsassociated risk of plaque rupture. Such agents may reduce or replace theneed for the use of cholesterol modifying drugs or HDL elevating drugs,with their attendant toxicities including, but not limited to, flushing,liver damage and muscle damage such as myopathy. Such agents may alsoreduce the need for, or prolong the period until, surgery is required toopen the vessel wall or until use of anticoagulants is required to limitdamage due to potential plaque rupture.

Labeled Compounds and Assay Methods

Another aspect of the present invention relates to fluorescent dye, spintable, heavy metal or radio-labeled compounds of Formula I that would beuseful not only in imaging but also in assays, both in vitro and invivo, for localizing and quantitating the chemokine receptor in tissuesamples, including human, and for identifying chemokine receptor ligandsby inhibition binding of a labeled compound. Accordingly, the presentinvention includes chemokine receptor assays that contain such labeledcompounds.

The present invention further includes isotopically-labeled compounds ofFormula I. An “isotopically” or “radio-labeled” compound is a compoundof the invention where one or more atoms are replaced or substituted byan atom having an atomic mass or mass number different from the atomicmass or mass number typically found in nature (i.e., naturallyoccurring). Suitable radionuclides that may be incorporated in compoundsof the present invention include but are not limited to ²H (also writtenas D for deuterium), ³H (also written as T for tritium), ¹¹C, ¹³C, ¹⁴C,¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I,¹²⁴I, ¹²⁵I and ¹³¹I. The radionuclide that is incorporated in theinstant radio-labeled compounds will depend on the specific applicationof that radio-labeled compound. For example, for in vitro chemokinereceptor labeling and competition assays, compounds that incorporate ³H,¹⁴C, ⁸²Br, ¹²⁵I, ¹³¹I, ³⁵S or will generally be most useful. Forradio-imaging applications ¹¹C, ¹⁸F, ¹²⁵I, ¹²³I, ¹²⁴I, ¹³¹I, ⁷⁵Br, ⁷⁶Bror ⁷⁷Br will generally be most useful.

It is understood that a “radio-labeled” or “labeled compound” is acompound that has incorporated at least one radionuclide. In someembodiments the radionuclide is selected from the group consisting of³H, ¹⁴C, ¹²⁵I, ³⁵S and ⁸²Br.

Synthetic methods for incorporating radio-isotopes into organiccompounds are applicable to compounds of the invention and are wellknown in the art A radio-labeled compound of the invention can be usedin a screening assay to identify/evaluate compounds. In general terms, anewly synthesized or identified compound (i.e., test compound) can beevaluated for its ability to reduce binding of the radio-labeledcompound of the invention to the chemokine receptor. Accordingly, theability of a test compound to compete with the radio-labeled compoundfor binding to the chemokine receptor directly correlates to its bindingaffinity.

Kits

The present invention also includes pharmaceutical kits useful, forexample, in the treatment or prevention of chemokine-associated diseaseswhich include one or more containers containing a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula I. Such kits can further include, if desired, one or more ofvarious conventional pharmaceutical kit components, such as, forexample, containers with one or more pharmaceutically acceptablecarriers, additional containers, etc., as will be readily apparent tothose skilled in the art. Instructions, either as inserts or as labels,indicating quantities of the components to be administered, guidelinesfor administration, and/or guidelines for mixing the components, canalso be included in the kit.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of noncriticalparameters which can be changed or modified to yield essentially thesame results.

EXAMPLES Example 1 Preparation ofN-[(1R,3S)-3-isopropyl-3-({4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}carbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine

Step A-1

4,4-Dimethoxytetrahydro-2H-pyran-3-ol

To a solution of 4-methoxy-3,6-dihydro-2H-pyran (5.00 g, 43.8 mmol) inmethanol (100 mL) at 0° C. was dropwise added a solution ofm-chloroperbenzoic acid (15.1 g, 87.6 mmol) in methanol (15 mL). Afterbeing stirred for 5 h, methanol was removed in vacuo and the whiteresidue was dissolved in methylene chloride (300 mL). To the solutionwas added K₂CO₃. The resulting solution was stirred for 1 h and filteredthrough celite. The filtrate was evaporated in vacuo to provide thedesired product which was used directly for the next reaction withoutpurification.

Step A-2

3,4,4-Trimethoxytetrahydro-2H-pyran

To a solution of 4,4-dimethoxytetrahydro-2H-pyran-3-ol (6.00 g, 37.0mmol) in THF (100 mL) at 0° C. was added sodium hydride (1.48 g, 37.0mmol). After being stirred at 0° C. for 1 h, methyl iodide (4.61 mL,74.0 mmol) was added dropwise. The reaction was allowed to warm up toambient temperature and quenched using aqueous NH₄Cl. The product wasextracted with ether three times. The combined extracts were dried overNa₂SO₄ and concentrated. Purification by flash chromatography on silicagel (10% ether to 60% ether/hexanes) provided the desired product. ¹HNMR (CDCl₃) δ 4.05-3.95 (1H, m), 3.80-3.70 (1H, m), 3.60-3.50 (3H, m),3.50 (3H, s), 3.30 (3H, s), 3.10 (3H, s), 2.00-1.70 (2H, m).

Step A-3

3-Methoxytetrahydro-4H-pyran-4-one

To a solution of 3,4,4-trimethoxytetrahydro-2H-pyran (4 g, 20 mmol) inTHF/H₂O (60 mL/10 mL) was added concentrated HCl (6 mL). After beingstirred for 1 h, THF was removed in vacuo. The aqueous solution wasextracted with ether (3×100 mL). The extracts were dried andconcentrated in vacuo to provide the desired product. ¹H NMR (CDCl₃) δ4.30-4.10 (2H, m), 3.75-3.65 (2H, m), 3.60-3.50 (1H, m), 3.50 (3H, s),2.70-2.50 (2H, m).

Step B-1

Methyl(1R,4S)-4-[(tert-Butoxycarbonyl)amino]cyclopent-2-ene-1-carboxylate

To a solution of(1R,4S)-4-[(tert-butoxycarbonyl)amino]cyclopent-2-ene-1-carboxylic acid(10.0 g, 44 mmol) in DMF (25 mL) was added potassium carbonate (6.33 g,45.8 mmol) followed by methyl iodide (4.0 mL, 64 mmol). After beingstirred at room temperature overnight, the reaction mixture was dilutedwith EtOAc. The solution was washed with water four times and brine onetime, dried (MgSO₄) and concentrated. The residue was dried under highvacuum overnight to provide the title compound (11 g, 99%). MScalculated for C₁₂H₁₉NO₄: (M+H)⁺ 242; found 142.1 (M-Boc+H)⁺. ¹H NMR(CDCl₃) δ 5.86 (m, 2H), 4.90 (m, 1H), 4.80 (m, 1H), 3.72 (s, 3H), 3.50(m, 1H), 2.51 (m, 1H), 1.86 (m, 1H), 1.42 (s, 9H).

Step B-2

Methyl(1S,4S)-4-[(tert-Butoxycarbonyl)amino]-1-isopropylcyclopent-2-ene-1-carboxylate

To a 1.00 M solution of lithium hexamethyldisilazide in THF (202 mL) at−78° C. was added a solution of methyl(1R,4S)-4-[(tert-butoxycarbonyl)amino]cyclopent-2-ene-1-carboxylate(22.10 g, 91.59 mmol) in THF (36.2 mL) over 10 min. The solution wasstirred at −78° C. for 30 min before isopropyl iodide (10.0 mL, 100mmol) was added in one portion. The mixture was then moved to a freezerreading at −24° C. and kept overnight. The reaction was quenched withaqueous ammonium chloride and the resulting solution was extracted withether three times. The ether layers were dried over sodium sulfate andevaporated in vacuo. The residue was purified by flash chromatography onsilica eluting with 10% ethyl acetate/hexane to give the title compound(20.2 g). MS calculated for C₁₅H₂₅NO₄: (M+H)⁺ 284; found 184.2(M-Boc+H)⁺.

Step B-3

(1S,4S)-4-[(tert-Butoxycarbonyl)amino]-1-isopropylcyclopent-2-ene-1-carboxylicAcid

To a solution of methyl(1S,4S)-4-[(tert-butoxycarbonyl)amino]-1-isopropylcyclopent-2-ene-1-carboxylate(18.42 g, 65 mmol) in THF (500 mL), methanol (500 mL) and water (100 mL)was added lithium hydroxide monohydrate (5.00 g, 119 mmol). The mixturewas heated to reflux overnight. After 18 hours, TLC indicated a verytrace amount of starting material. The organic solvents were removed invacuo and the aqueous layer was extracted with ether (200 mL) to removethe unreacted starting material. The aqueous layer was acidified withconcentrated HCl to pH=4 while being cooled in an ice bath. Theresulting solution was extracted with methylene chloride three times.The extracts were dried over MgSO₄ and concentrated to give a solid (17g). The solid was dissolved in hot ethyl acetate (22 mL) and hexanes(550 mL) were added to the solution. The solution was slowly cooled downto room temperature before putting into a freezer reading at −22 to −24°C. After two days, the crystals were removed off and the liquid wasevaporated in vacuo to give the desired product as a white foamy solid(9.78 g, 56%). MS calculated for C₁₄H₂₃NO₄: (M+H)⁺ 270; found 170.1(M-Boc+H)⁺.

Step B-4

(1S,3R)-3-[(tert-Butoxycarbonyl)amino]-1-isopropylcyclopentanecarboxylicAcid

To a solution of(1S,4S)-4-[(tert-butoxycarbonyl)amino]-1-isopropylcyclopent-2-ene-1-carboxylicacid (9.78 g, 36.3 mmol) in ethanol (250 mL) was added 10% palladium oncarbon (550 mg). The mixture was shaken under hydrogen at 55 psiovernight and filtered through celite. The filtrate was evaporated invacuo to afford the title compound (9.45 g, 96%). MS calculated forC₁₄H₂₅NO₄: (M+H)⁺ 272; found 172.1 (M-Boc+H)⁺.

Step C

tert-Butyl[(1R,3S)-3-(4-[3-(trifluoromethyl)phenyl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate

(1S,3R)-3-[(tert-Butoxycarbonyl)amino]-1-isopropyleyclopentanecarboxylicacid (0.10 g, 0.37 mmol), N-[3-Trifluoromethyl]phenylpiperazine (85 mg,0.37 mmol), triethylamine (0.10 mL, 0.74 mmol) andbenzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate(0.16 g, 0.37 mmol) were mixed in methylene chloride (5 mL) and stirredat room temperature overnight. The reaction mixture was diluted withEtOAc and washed with saturated NaHCO₃. The aqueous layer was extractedwith EtOAc three times. The combined organic layers were dried (MgSO₄),concentrated and flash chromatographed (50% EtOAc/Hex to EA) to give thedesired product (86 mg, 53%). MS calculated for C₂₅H₃₆F₃N₃O₃: (M+H)484.3; found 384.2 (M+H-Boc).

Step D

(1R,3S)-3-(4-[3-(Trifluoromethyl)phenyl]piperazin-1-ylcarbonyl)cyclopentanaminebis(trifluoroacetate)

tert-Butyl[(1R,3S)-3-(4-[3-(trifluoromethyl)phenyl]-piperazin-1-ylcarbonyl)-cyclopentyl]-carbamate(82 mg, 0.18 mmol) was treated with trifluoroacetic Acid (3 mL, 0.04mol) in methylene chloride (3 mL) at room temperature for 1 h. Themixture was concentrated to provide the desired product (98 mg, 93%). MScalculated for C₂₀H₂₈F₃N₃O: (M+H) 384.3; found 384.2.

Step E

N-[(1R,3S)-3-Isopropyl-3-({4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}carbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine

To a solution of(1R,3S)-3-isopropyl-3-({4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}carbonyl)cyclopentanaminebis(trifluoroacetate) (140 mg, 0.23 mmol),3-methoxytetrahydro-4H-pyran-4-one (90 mg, 0.69 mmol) and triethylamine(0.096 mL, 0.69 mmol) in methylene chloride (10 mL) was added sodiumtriacetoxyborohydride (97 mg, 0.46 mmol). After being stirred at roomtemperature overnight, the reaction mixture was diluted with EtOAc andwashed with saturated Na₂CO₃. The aqueous layer was extracted with EtOActhree times. The combined organic layers were dried (MgSO₄),concentrated and purified on silica gel eluting with EtOAc to 1%Et₃N/EtOAc affording 105 mg (92%) of the desired product. The productwas separated by chiral HPLC to give two major isomers. MS calculatedfor C₂₆H₃₈F₃N₃O₃: (M+H) 498; found 498.2 for both isomers.

Example 2

Preparation of3-ethoxy-N-[(1R,3S)-3-isopropyl-3-({4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}carbonyl)cyclopentyl]tetrahydro-2H-pyran-4-amineStep A-1

3-Ethoxy-4,4-dimethoxytetrahydro-2H-pyran

To a solution of 4,4-dimethoxytetrahydro-2H-pyran-3-ol (2.0 g, 12 mmol)in THF (20 mL) cooled in an ice bath was slowly added sodium hydride(0.60 g, 15 mmol) and the resulting slurry was stirred for 1 h.Iodoethane (1.5 mL, 19 mmol) was added and the mixture was stirred atroom temperature overnight. More sodium hydride (0.6 g) and iodoethane(3 mL) were added and stirring was continued another overnight. Thereaction was quenched with water. The resulting solution was extractedwith EtOAc twice and ether twice. The combined extracts were dried,concentrated and purified on silica gel (20% EtOAc/hexanes) to give 2.1g (90%) of desired product. ¹H NMR (CDCl₃) δ 3.32-4.00 (7H, m), 3.30(3H, s), 3.20 (3H, s), 2.05-1.70 (2H, m), 1.25-1.22 (3H, m).

Step A-2

3-Ethoxytetrahydro-4H-pyran-4-one

To a solution of 3-ethoxy-4,4-dimethoxytetrahydro-2H-pyran (2.1 g, 11mmol) in THF/water (50 mL/10 mL) was added concentrated HCl (6 mL).After being stirred at room temperature for 1 h, the mixture was dilutedwith EtOAc. The organic layer was separated and the aqueous layer wasextracted with ether five times. The combined organic layers were driedover MgSO₄, filtered and concentrated to give 1.55 g (97%) of thedesired product ¹H NMR (CDCl₃) δ 4.30-3.50 (7H, m), 2.62-2.57 (2H, m),1.30-1.20 (3H, t, J=5 Hz).

Step B

3-Ethoxy-N-[(1R,3S)-3-isopropyl-3-({4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}carbonyl)cyclopentyl]tetrahydro-2H-pyran-4-amine

To a solution of(1R,3S)-3-isopropyl-3-({4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}carbonyl)cyclopentanaminedihydrochloride (200 mg, 0.438 mmol), 3-ethoxytetrahydro-4H-pyran-4-one(130 mg, 0.88 mmol) and triethylamine (0.18 mL, 1.3 mmol) in methylenechloride (10 mL) was added sodium triacetoxyborohydride (180 mg, 0.88mmol). After being stirred at room temperature overnight, the reactionmixture was diluted with EtOAc and washed with saturated Na₂CO₃. Theaqueous layer was extracted with EtOAc three times. The combined organiclayers were dried (MgSO₄), concentrated and purified on silica gel(EtOAc to 1% Et₃N/EtOAc to 5% Et₃N/EtOAc) affording 230 mg of product.The product was separated by chiral HPLC to give two major isomers:isomer 1 (110 mg) and isomer 2 (77 mg). MS calculated for C₂₇H₄₀F₃N₃O₃:(M+H) 512; found 512.2.

Example 3

Preparation ofN-[(1R,3S)-3-isopropyl-3-({4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}carbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amineStep A

1-[4-(Trifluoromethyl)pyridin-2-yl]piperazine

A solution of 2-chloro-4-(trifluoromethyl)pyridine (2.0 g, 11 mmol),piperazine (3 g, 30 mmol) and triethylamine (3.1 mL, 22 mmol) in DMF (10mL) was heated at 100° C. overnight and concentrated in vacuo. Theresidue was purified by column chromatography on silica gel (EtOAc toEtOAc/MeOH/Et₃N=9/1/0.5) to give 1.09 g (43%) of pure product MScalculated for C₁₀H₁₂F₃N₃: (M+H) 232; found 232.1.

Step B

tert-Butyl[(1R,3S)-3-isopropyl-3-({4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}carbonyl)cyclopentyl]carbamate

To a solution of 1-[4-(trifluoromethyl)pyridin-2-yl]piperazine (145 mg,0.627 mmol),(1S,3R)-3-[(tert-butoxycarbonyl)amino]-1-isopropylcyclopentanecarboxylicacid (140 mg, 0.52 mmol) in methylene chloride (10 mL) was addedbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(253 mg, 0.572 mmol) followed by triethylamine (0.156 mL, 1.12 mmol).After being stirred overnight, the reaction mixture was diluted withEtOAc and washed with saturated NaHCO₃. The aqueous layer was extractedwith EtOAc three times. The combined organic layers were dried (MgSO₄),concentrated and purified by flash chromatography on silica gel (20%EtOAc/hexanes to 40% EtOAc/hexanes) to give 0.15 g of desired product.MS calculated for C₂₄H₃₅F₃N₄O₃: (M+H) 485; found 385.2 (M-Boc+H).

Step C

(1R,3S)-3-Isopropyl-3-({4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}carbonyl)cyclopentanamine

tert-Butyl[(1R,3S)-3-isopropyl-3-({4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}carbonyl)cyclopentyl]carbamate(150 mg, 0.31 mmol) was treated with a 4.0 M solution of HCl in1,4-dioxane (10 mL) at room temperature for 1 hr and concentrated atreduced pressure to give the product which was used for next stepwithout purification. MS calculated for C₁₉H₂₇F₃N₄O: (M+H) 385; found385.2.

Step D

N-[(1R,3S)-3-Isopropyl-3-({4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl})carbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine

To a solution of(1R,3S)-3-isopropyl-3-({4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}carbonyl)cyclopentanaminedihydrochloride (140 mg, 0.31 mmol), 3-methoxytetrahydro-4H-pyran-4-one(80 mg, 0.61 mmol) and triethylamine (0.21 mL, 1.5 mmol) in methylenechloride (10 mL) was added sodium triacetoxyborohydride (190 mg, 0.92mmol). After being stirred overnight, the reaction mixture was dilutedwith EtOAc and washed with saturated Na₂CO₃. The aqueous layer wasextracted with EtOAc three times. The combined organic layers were dried(MgSO₄), concentrated and purified by flash chromatography on silica gel(EtOAc to 1% Et₃N/EtOAc to 5% Et₃N/EtOAc) to give 101 mg of product. Theproduct was further separated by chiral HPLC to give isomer 1 (55 mg)and isomer 2 (37 mg). LCMS calculated for C₂₅H₃₇F₃N₄O₃ (M+1) 499; found499.2 for both isomers.

Example 4 Preparation ofN-[(1R,3S)-3-isopropyl-3-({4-[5-(trifluoromethyl)pyridin-3-yl]piperazin-1-yl}carbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine

Step A-1

3-Bromo-5-iodopyridine

To a solution of 3,5-dibromopyridine (48 g, 200 mmol) in THF (200 mL)was added a 2 M solution of isopropylmagnesium chloride in THF (80 mL).After being stirred at room temperature for 2 h, the solution was cooledto −78° C. To it was added a precooled solution of iodine (51 g, 200mmol) in THF (100 mL). The mixture was diluted with ether and washedwith a saturated solution of ammonium chloride, a 2 M solution of sodiumthiosulfate, and brine. The resulting organic layer Was dried overMgSO₄, filtered and concentrated. Crystallization from ethanol gave 33.5g (58%) of desired product, ¹H NMR (CDCl₃) δ 8.75 (1H, s), 8.60 (1H, s),8.20 (1H, s).

Step A-2

tert-Butyl4-(5-bromopyridin-3-yl)piperazine-1-carboxylate

A solution of 3-bromo-5-iodopyridine (13.0 g, 45.8 mmol), tert-butylpiperazine-1-carboxylate (8.53 g, 45.8 mmol), copper(I) iodide (0.871 g,4.57 mmol), K₃PO₄ (19.46 g, 91.68 mmol), 1,2-ethanediol (5.1 mL, 91mmol) in isopropyl alcohol (80 mL) in a sealed tube was heated at 80° C.in an oil bath for 2 days. After cooling to room temperature, thereaction mixture was filtered through celite. The filtrate wasconcentrated in vacuo. The residue was taken up in EtOAc and thesolution was washed with saturated NaHCO₃, dried (MgSO₄) andconcentrated. Purification by flash chromatography on silica gel (20%EtOAc/hexanes to 30% EtOAc/hexanes) afforded 5.75 g (37%) of desiredproduct. MS calculated for C₁₄H₂₀BrN₃O₂: (M+H) 343; found 342.0, 344.0.

Step A-3

tert-Butyl4-(5-iodopyridin-3-yl)piperazine-1-carboxylate

To a solution of tert-butyl 4-(3-bromophenyl)piperazine-1-carboxylate(2.0 g, 5.9 mmol) in THF (20 mL) was added a 2 M solution ofisopropylmagnesium chloride in THF (5 mL). After being stirred at roomtemperature for 2 h, the solution was cooled to −78° C. To it was addeda precooled solution of iodine (3.0 g, 12 mmol) in THF (2 mL). Afterbeing stirred at −78° C. for 30 min and at room temperature for another30 min, the mixture was diluted with ethyl acetate, washed withsaturated ammonium chloride, 2 M solution of sodium thiosulfate andbrine, dried (MgSO₄) and concentrated. The residue was purified by flashchromatography on silica gel (20% EtOAc/hexanes to 50% EtOAc/hexanes) togive the desired product (1.40 g) in 75% purity. MS calculated forC₁₅H₂₁IN₂O₂: (M+H) 390; found 390.0.

Step A-4

tert-Butyl4-[5-(trifluoromethyl)pyridin-3-yl]piperazine-1-carboxylate

Copper(I) iodide (0.49 g, 2.6 mmol) and potassium fluoride (0.15 g, 2.6mmol) in a flask were flame-heated under gentle shaking and at highvacuum until a greenish color appeared. A solution of tert-butyl4-(3-iodophenyl)piperazine-1-carboxylate (0.5 g, 1.0 mmol) and(trifluoromethyl)trimethylsilane (0.37 g, 2.6 mmol) in DMF (5 mL) wasadded. The brown solution was stirred at room temperature overnight.More (trifluoromethyl)trimethylsilane (0.37 g) was added. The mixturewas heated at 50° C. overnight, diluted with EtOAc and washed withsaturated ammonium chloride. The aqueous layer was extracted with EtOActhree times. The combined organic layers were dried (MgSO₄),concentrated and purified by flash chromatography on silica gel (20% to40% EtOAc/hexanes) to give 120 mg of desired product. MS calculated forC₁₅H₂₀F₃N₃O₂: (M+H) 332; found 332.1.

Step A-5

1-[5-(Trifluoromethyl)pyridin-3-yl]piperazine

tert-Butyl 4-[5-(trifluoromethyl)pyridin-3-yl]piperazine-1-carboxylate(0.24 g, 0.25 mmol) was treated with a 4.0 M solution of HCl in1,4-dioxane (7 mL) at room temperature for 1 h and concentrated. Theresidue was used for next step without further purification. MScalculated for C₁₀H₁₂F₃N₃: (M+H) 232; found 232.1.

Step B

tert-Butyl[(1R,3S)-3-isopropyl-3-({4-[5-(trifluoromethyl)pyridin-3-yl]piperazin-1-yl}carbonyl)cyclopentyl]carbamate

To a solution of 1-[5-(trifluoromethyl)pyridin-3-yl]piperazinetrihydrochloride (0.22 g, 0.23 mmol),(1S,3R)-3-[(tert-butoxycarbonyl)amino]-1-isopropylcyclopentanecarboxylicacid (0.18 g, 0.66 mmol) in methylene chloride (10 mL) was addedbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(0.338 g, 0.764 mmol) followed by triethylamine (0.22 mL, 1.6 mmol). Themixture was stirred at room temperature overnight and diluted withEtOAc. The solution was washed with saturated NaHCO₃, dried (MgSO₄) andconcentrated. The residue was purified by flash chromatography on silicagel (20% EtOAc/hexanes to 40% EtOAc/hexanes) to give 0.19 g (61%) ofdesired product. MS calculated for C₂₄H₃₅F₃N₄O₃: (M+H) 485; found 485.2.

Step C

(1R,3S)-3-Isopropyl-3-({4-[5-(trifluoromethyl)pyridin-3-yl]piperazin-1-yl}carbonyl)cyclopentanamine

tert-Butyl[(1R,3S)-3-isopropyl-3-({4-[5-(trifluoromethyl)pyridin-3-yl]piperazin-1-yl}carbonyl)cyclopentyl]carbamate(190 mg, 0.14 mmol) was treated with a 4.0 M solution of HCl in1,4-dioxane (5 mL) for 1 h at room temperature. The mixture wasconcentrated and purified by HPLC to provide 35 mg of desired product.MS calculated for C₁₉H₂₇F₃N₄O; (M+H) 385; found 385.1.

Step D

N-[(1R,3S)-3-Isopropyl-3-({4-[5-(trifluoromethyl)pyridin-3-yl]piperazin-1-yl}carbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine

To a solution of(1R,3S)-3-isopropyl-3-({4-[5-(trifluoromethyl)pyridin-3-yl]piperazin-1-yl}carbonyl)cyclopentanaminetris(trifluoroacetate) (25 mg, 0.034 mmol),3-methoxytetrahydro-4H-pyran-4-one (13 mg, 0.10 mmol) and triethylamine(0.024 mL, 0.17 mmol) in methylene chloride (5 mL) was added sodiumtriacetoxyborohydride (22 mg, 0.10 mmol). The mixture was stirred underN₂ at room temperature overnight and diluted with EtOAc. The resultingsolution was washed with saturated NaHCO₃, dried (MgSO₄), andconcentrated. The residue was purified by flash chromatography on silicagel (EtOAc to EtOAc/MeOH/Et₃N=9:1:0.5) to give 14 mg of desired productas a mixture of two isomers. The two isomers were separated by chiralHPLC to give peak 1 (6.6 mg) and peak 2 (4.7 mg). LCMS calculated forC₂₅H₃₇F₃N₄O₃: (M+1) 499; found 499.2 for both isomers.

Example 5 Preparation ofN-{(1R,3S)-3-isopropyl-3-[(4-phenyl-3,6-dihydropyridin-1(2H)-yl)carbonyl]cyclopentyl}-3-methoxytetrahydro-2H-pyran-4-amine

Step A

tert-Butyl{(1R,3S)-3-isopropyl-3-[(4-phenyl-3,6-dihydropyridin-1(2H)-yl)carbonyl]cyclopentyl}carbamate

In a dried flask,(1S,3R)-3-[(tert-butoxycarbonyl)amino]-1-isopropyl-cyclopentanecarboxylicacid (200 mg, 0.7 mmol) and 4-phenyl-1,2,3,6-tetrahydropyridine (160 mg,0.81 mmol) were suspended in methylene chloride (4 mL) under N₂.Triethylamine (0.22 g, 2.2 mmol) was added followed bybenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(0.36 g, 0.81 mmol). The reaction was stirred overnight at roomtemperature and quenched by addition of saturated NaHCO₃ solution. Theresulting solution was extracted with methylene chloride three times.The combined extracts were dried (MgSO₄), filtered and concentrated.Purification by flash chromatography on silica gel (gradient: 0-45% Bover 15 min. Bottle A=hexanes, bottle B=EtOAc) to give 234 mg (80%) ofdesired product. MS calculated for C₂₅H₃₆N₂O₃: (M+H) 413; found 413.2.

Step B

(1R,3S)-3-Isopropyl-3-[(4-phenyl-3,6-dihydropyridin-1(2H)-yl)carbonyl]cyclopentanamine

tert-Butyl{(1R,3S)-3-isopropyl-3-[(4-phenyl-3,6-dihydropyridin-1(2H)-yl)carbonyl]-cyclopentyl}carbamate(0.23 g, 0.56 mmol) was dissolved in a 1.0 M solution of HCl in ether (4mL). After being stirred at room temperature for 2 h, the solution wasconcentrated to give a colorless oil (170 mg). MS calculated forC₂₀H₂₈N₂O: (M+H) 313; found 313.2.

Step C N-{(1R,3S)-3-Isopropyl-3-[(4-phenyl-3,6-dihydropyridin-1(2H)-yl)carbonyl]-cyclopentyl}tetrahydro-2H-pyran-4-amine

To a solution of(1R,3S)-3-isopropyl-3-[(4-phenyl-3,6-dihydropyridin-1(2H)-yl)carbonyl]cyclopentanaminehydrochloride (50 mg, 0.1 mmol), 3-methoxytetrahydro-4H-pyran-4-one (56mg, 0.43 mmol), and triethylamine (0.07 mL, 0.5 mmol) in methylenechloride (2 mL) was added sodium triacetoxyborohydride (91 mg, 0.43mmol). After being stirred at room temperature overnight, saturatedNaHCO₃ was added. The solution was extracted with methylene chloridethree times. The combined extracts were dried (MgSO₄), filtered, andconcentrated. Purification by flash chromatography on silica gel(gradient 0-15% B over 15 min. Bottle A=1% NH₄OH/3% MeOH/EtOAc, BottleB=1% NH₄OH/MeOH) afforded the desired compound. MS calculated forC₂₆H₃₅N₂O₃: (M+H) 427; found 427.3.

Example 6 Preparation of1-({(1S,3R)-1-isopropyl-3-[(3-methoxytetrahydro-2H-pyran-4-yl)amino]cyclopentyl}carbonyl)-4-phenylpiperidin-4-ol

Step A

tert-Butyl{(1R,3S)-3-[(4-hydroxy-4-phenylpiperidin-1-yl)carbonyl]-3-isopropylcyclopentyl}carbamate

To a solution of(1S,3R)-3-[(tert-butoxycarbonyl)amino]-1-isopropylcyclopentane-carboxylicacid (200 mg, 0.7 mmol), 4-phenylpiperidin-4-ol (140 mg, 0.81 mmol), andtriethylamine (0.15 g, 1.5 mmol) in methylene chloride (4 mL) was added(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate(0.42 g, 0.81 mmol). After being stirred overnight at room temperature,the reaction was quenched by addition of a saturated NaHCO₃ solution.The resulting solution was extracted with methylene chloride threetimes. The combined extracts were dried (MgSO₄), filtered, andconcentrated. The crude was carried on to the next step withoutpurification. MS calculated for C₂₅H₃₈N₂O₄: (M+H) 431; found 431.2.

Step B

1-{[(1S,3R)-3-Amino-1-isopropylcyclopentyl]carbonyl}-4-phenylpiperidin-4-ol

tert-Butyl{(1R,3S)-3-[(4-hydroxy-4-phenylpiperidin-1-yl)carbonyl]-3-isopropyl-cyclopentyl}carbamate(0.30 g, 0.70 mmol) was dissolved in a 2.0 M solution of HCl in ether(10 mL). After being stirred for 3.5 h, a few drops of MeOH was added toget a clear solution. The mixture was concentrated to give an oil. Thecrude product was used in the next reaction without purification. MScalculated for C₂₀H₃₀N₂O₂: (M+H) 331; found 331.2.

Step C

1-({(1S,3R)-1-Isopropyl-3-[(3-methoxytetrahydro-2H-pyran-4-yl)amino]cyclopentyl}carbonyl)-4-phenylpiperidin-4-ol

To a solution of1-{[(1S,3R)-3-amino-1-isopropylcyclopentyl]carbonyl}-4-phenylpiperidin-4-olhydrochloride (50 mg, 0.1 mmol), 3-methoxytetrahydro-4H-pyran-4-one (53mg, 0.41 mmol), and triethylamine (0.066 mL, 0.48 mmol) in methylenechloride (2 mL) was added sodium triacetoxyborohydride (0.087 g, 0.41mmol). After being stirred overnight at room temperature, the reactionwas quenched with a saturated NaHCO₃ solution. The resulting solutionwas extracted with methylene chloride three times. The combined extractswere dried (MgSO₄), filtered, and concentrated. Purification by flashchromatography on silica gel (0-20% B over 17 min. Bottle A=1% NH₄OH/2%MeOH/EtOAc, Bottle B=1% NH₄OH/MeOH) afforded the desired product. MScalculated for C₂₆H₄₀N₂O₄: (M+H) 445; found 445.2.

Example 7 Preparation of1-({(1S,3R)-1-isopropyl-3-[(3-methoxytetrahydro-2H-pyran-4-yl)amino]cyclopentyl})carbonyl)-4-[2-(trifluoromethyl)phenyl]piperidin-4-ol

Step A-1

tert-Butyl4-hydroxy-4-[2-(trifluoromethyl)phenyl]piperidine-1-carboxylate

To a solution of 1-bromo-2-(trifluoromethyl)benzene (1.18 g, 5.24 mmol)in THF (20 mL) cooled at −78° C. was dropwise added a 1.60 M solution ofn-butyllithium in hexane (3.4 mL). After being stirred for 40 min; asolution of tert-butyl 4-oxo-1-piperidinecarboxylate (1.0 g, 5.0 mmol)in THF (3 mL) was added and the solution stirred for 1 h at −78° C. Thereaction was quenched with saturated ammonium chloride. The resultingsolution was extracted with methylene chloride three times. The combinedextracts were dried (MgSO₄), filtered, and concentrated to give 0.78 gof a white solid which was used for the next reaction withoutpurification. MS calculated for C₁₇H₂₂F₃NO₃: (M+H) 346; found 246.0(M-Boc+1).

Step A-2

4-[2-(Trifluoromethyl)phenyl]piperidin-4-ol

tert-Butyl4-hydroxy-4-[2-(trifluoromethyl)phenyl]piperidine-1-carboxylate (0.40 g,1.0 mmol) was dissolved in a 2.0 M solution of HCl in ether (5 mL).After being stirred at room temperature overnight, the solution wasdiluted with ether. The white solid was filtered and washed with etherto give 170 mg of pure product. MS calculated for C₁₂H₁₄F₃NO: (M+H) 246;found 246.1.

Step B

tert-Butyl[(1R,3S)-3-({4-Hydroxy-4-[2-(trifluoromethyl)phenyl]piperidin-1-yl}carbonyl)-3-isopropylcyclopentyl]carbamate

To a solution of(1S,3R)-3-[(tert-butoxycarbonyl)amino]-1-isopropylcyclopentanecarboxylicacid (150 mg, 0.55 mmol), 4-[2-(trifluoromethyl)phenyl]piperidin-4-olhydrochloride (170 mg, 0.60 mmol), and triethylamine (0.17 g, 1.6 mmol)in methylene chloride (3 mL) was added(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate(0.31 g, 0.60 mmol). After being stirred for 2.5 h, the reaction wasquenched by addition of a saturated NaHCO₃ solution. The resultingsolution was extracted with methylene chloride three times. The combinedextracts were dried (MgSO₄), filtered, and concentrated. The crudeproduct was carried on to the next step without purification. MScalculated for C₂₆H₃₇F₃N₂O₄: (M+H) 499; found 499.2.

Step C

1-{[(1S,3R)-3-Amino-1-isopropylcyclopentyl]carbonyl}-4-[2-(trifluoromethyl)phenyl]piperidin-4-ol

To a flask containing tert-butyl[(1R,3S)-3-({4-hydroxy-4-[2-(trifluoromethyl)phenyl]piperidin-1-yl}carbonyl)-3-isopropylcyclopentyl]carbamate(0.27 g, 0.54 mmol) was added a 2.00 M solution of HCl in ether (5 mL)and the resulting mixture stirred for 3.5 h. The solution wasconcentrated to give an oil which was used in the next reaction withoutpurification. MS calculated for C₂₁H₂₉F₃N₂O₂: (M+H) 399; found 399.2.

Step D

1-({(1S,3R)-1-Isopropyl-3-[(3-methoxytetrahydro-2H-pyran-4-yl)amino]cyclopentyl}carbonyl)-4-[2-(trifluoromethyl)phenyl]piperidin-4-ol

To a solution of1-{[(1S,3R)-3-amino-1-isopropylcyclopentyl]carbonyl}-4-[2-(trifluoromethyl)phenyl]piperidin-4-olhydrochloride (50 mg, 0.1 mmol), 3-methoxytetrahydro-4H-pyran-4-one (45mg, 0.34 mmol), and triethylamine (0.048 mL, 0.34 mmol) in methylenechloride (2 mL) was added sodium triacetoxyborohydride (0.073 g, 0.34mmol). After being stirred overnight at room temperature, a saturatedsolution of NaHCO₃ was added. The solution was extracted with methylenechloride three times. The combined extracts were dried (MgSO₄),filtered, and concentrated. Purification by flash chromatography onsilica gel (0-20% B over 15 min. Bottle A=1% NH₄OH/2% MeOH/EtOAc, BottleB=1% NH₄OH/MeOH) afforded 15 mg of the desired product as an oil. MScalculated for C₂₇H₃₉F₃N₂O₄: (M+H) 513; found 513.2.

Example 8 Preparation of1-[((1S,3R)-1-isopropyl-3-{[3-methoxytetrahydro-2H-pyran-4-yl]amino}cyclopentyl)carbonyl]-4-[3-(trifluoromethyl)phenyl]piperidin-4-ol

The title compound was prepared using procedures analogous to thosedescribed for Example 7. MS calculated for C₂₇H₃₉F₃N₂O₄: (M+H) 513;found 513.2.

Example 9 Preparation of1-[((1S,3R)-1-isopropyl-3-{([3-methoxytetrahydro-2H-pyran-4-yl]amino}cyclopentyl)carbonyl]-4-[4-(trifluoromethyl)phenyl]piperidin-4-ol

The title compound was prepared using procedures analogous to thosedescribed for Example 21. MS calculated for C₂₇H₃₉F₃N₂O₄: (M+H) 513;found 513.2.

Example 10 Preparation ofN-((1R,3S)-3-isopropyl-3-{[4-[2-(trifluoromethyl)phenyl]-3,6-dihydropyridin-1(2H)-yl]carbonyl}cyclopentyl)-3-methoxytetrahydro-2H-pyran-4-amine

Step A-1

tert-Butyl4-[2-(trifluoromethyl)phenyl]-3,6-dihydropyridine-1(2H)-carboxylate

To a solution of tert-butyl4-hydroxy-4-[2-(trifluoromethyl)phenyl]piperidine-1-carboxylate (0.75 g,2.2 mmol) in pyridine (15 mL) cooled in an ice bath was slowly addedthionyl chloride (0.79 mL, 11 mmol) and the mixture was warmed to roomtemperature and stirred overnight (17 h). The reaction was quenched withice water. The resulting solution was extracted with methylene chloridethree times. The combined extracts were dried (MgSO₄), filtered, andconcentrated. Purification by flash chromatography on silica gel (0-40%B over 25 min. Bottle A=hexanes, Bottle B=EtOAc) gave 209 g of thedesired product as a solid. MS calculated for C₁₇H₂₀F₃NO₂: (M+H) 328;found 228.0 (M-Boc+H).

Step A-2

4-[2-(Trifluoromethyl)phenyl]-1,2,3,6-tetrahydropyridine

To a solution of tert-butyl4-[2-(trifluoromethyl)phenyl]-3,6-dihydropyridine-1(2H)-carboxylate (200mg, 0.61 mmol) in methylene chloride (5 mL) was added trifluoroaceticacid (2.5 mL). After being stirred at room temperature for 45 min, thesolution was concentrated to give an oil. MS calculated for C₁₂H₁₂F₃N:(M+H) 228; found 228.1.

Step B

tert-Butyl((1R,3S)-3-isopropyl-3-{[4-phenyl-2-(trifluoromethyl)-3,6-dihydropyridin-1(2H)-yl]carbonyl}cyclopentyl)carbamate

To a solution of(1S,3R)-3-[(tert-butoxycarbonyl)amino]-1-isopropylcyclopentanecarboxylicacid (115 mg, 0.424 mmol) and4-[2-(trifluoromethyl)phenyl]-1,2,3,6-tetrahydropyridinetrifluoroacetate (152 mg, 0.445 mmol) in methylene chloride (2 mL) wasadded triethylamine (0.21 g, 2.1 mmol) followed bybenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(210 mg, 0.47 mmol). After being stirred overnight at room temperature,the reaction was quenched with saturated NaHCO₃ solution. The resultingsolution was extracted with methylene chloride three times. The combinedextracts were dried (MgSO₄), filtered, and concentrated. Purification byflash chromatography on silica gel (0-50% B over 15 min. BottleA=hexanes, Bottle B=EtOAc) provided 174 mg of desired product as a whitesolid. MS calculated for C₂₆H₃₅F₃N₂O₃: (M+H) 481; found 481.1.

Step C

(1R,3S)-3-Isopropyl-3-{[4-[2-(trifluoromethyl)phenyl]-3,6-dihydropyridin-1(2H)-yl]carbonyl}cyclopentanamine

tert-Butyl((1R,3S)-3-isopropyl-3-{[4-[2-(trifluoromethyl)phenyl]-3,6-dihydropyridin-1(2H)-yl]carbonyl}cyclopentyl)carbamate(0.17 g, 0.00035 mol) was dissolved in a 2.0 M solution of HCl in ether(2.2 mL). After being stirred for 2 h at room temperature, the solutionwas concentrated to give 144 mg of desired product as a clear oil. MScalculated for C₂₁H₂₇F₃N₂O: (M+H) 381; found 381.1.

Step DN-((1R,3S)-3-Isopropyl-3-{([4-[2-(trifluoromethyl)phenyl]-3,6-dihydropyridin-1(2H)-yl]carbonyl}cyclopentyl)tetrahydro-2H-pyran-4-amine

To a solution of(1R,3S)-3-isopropyl-3-{[4-[2-(trifluoromethyl)phenyl]-3,6-dihydropyridin-1(2H)-yl]carbonyl}cyclopentanaminehydrochloride (46 mg, 0.11 mmol), 3-methoxy-tetrahydro-4H-pyran-4-one(56 mg, 0.43 mmol), and triethylamine (0.054 mL, 0.39 mmol) in methylenechloride (2 mL) was added sodium triacetoxyborohydride (70 mg, 0.33mmol). After being stirred overnight at room temperature, a saturatedNaHCO₃ solution was added. The resulting solution was extracted withmethylene chloride three times. The combined extracts were dried(MgSO₄), filtered, and concentrated. Purification by flashchromatography on silica gel (0-20% B over 15 min. Bottle A=1% NH₄OH/2%MeOH/EtOAc, Bottle B=1% NH₄OH/MeOH) provided the desired product. MScalculated for C₂₇H₃₇F₃N₂O₃: (M+H) 495; found 495.2.

Example 11 Preparation ofN-((1R,3S)-3-isopropyl-3-{[4-3-(trifluoromethyl)phenyl-3,6-dihydropyridin-1(2H)-yl]carbonyl}cyclopentyl)-3-methoxytetrahydro-2H-pyran-4-amine

The title compound was prepared in a manner analogous to that describedfor Example 10. MS calculated for C₂₇H₃₇F₃N₂O₃: (M+H) 495; found 495.2.

Example 12 Preparation of3-ethoxy-N-((1R,3S)-3-isopropyl-3-{[4-[3-(trifluoromethyl)phenyl]-3,6-dihydropyridin-1(2H)-yl]carbonyl}cyclopentyl)tetrahydro-2H-pyran-4-amine

The title compound was prepared in a manner analogous to that describedfor Example 10. MS calculated for C₂₈H₃₉F₃N₂O₃: (M+H) 509; found 509.1.

Example 13 Preparation ofN-((1R,3S)-3-isopropyl-3-{[4-(trifluoromethyl)-3′,6′-dihydro-2,4′-bipyridin-1′(2′H)-yl]carbonyl}cyclopentyl)-3-methoxytetrahydro-2H-pyran-4-amine

Step A-1

2-Bromo-4-(trifluoromethyl)pyridine

A mixture of 2-chloro-4-(trifluoromethyl)pyridine (2.70 g, 14.9 mmol)and bromotrimethylsilane (3.90 mL, 29.6 mmol) in propanenitrile (15.0mL) was heated under reflux for 22 h. The product (very volatile) wascarefully rotary evaporated to give 4.07 g (propanenitrile contained) ofthick light brown suspension w/o further purification. LC-MS calculatedfor C₆H₃BrF₃N (M+H) 226.9; found 225.9/227.8.

Step A-2

tert-Butyl4-hydroxy-4-[4-(trifluoromethyl)pyridin-2-yl]piperidine-1-carboxylate

To a slightly cloudy solution of 2-bromo-4-(trifluoromethyl)pyridine(4.0 g, 14.2 mmol) in dry methylene chloride (52.7 mL) cooled at −78° C.was added a 1.6 M solution of n-butyllithium in hexanes (9.65 mL). Afterbeing stirred for 40 min at −78° C., a solution of tert-butyl4-oxo-1-piperidinecarboxylate (2.59 g, 12.9 mmol) in dry methylenechloride (10.0 mL) was added dropwise. The reaction was stirred at −78°C. for 1 h and quenched with aqueous NH₄Cl. THF was removed by rotaryevaporation. The aqueous layer was extracted with methylene chloridethree times. The combined organic layers were dried, filtered andconcentrated. The residue was purified by column chromatography onsilica gel (30:70 EtOAc/hexanes) to give 2.63 g (59%) of desired productas a brown oil. LC-MS calculated for C₁₆H₂₁F₃N₂O₃: (M+H) 347; found247.0 (M-Boc+1).

Step A-3

tert-Butyl4-(trifluoromethyl)-3′,6′-dihydro-2,4′-bipyridine-1′(2′H)-carboxylate

To a solution of tert-butyl4-hydroxy-4-[4-(trifluoromethyl)pyridin-2-yl]piperidine-1-carboxylate(2.00 g, 2.31 mmol) in pyridine (15.9 mL) cooled in an ice bath wasslowly added thionyl chloride (0.84 mL, 12 mmol). The mixture wasallowed to warm to room temperature and stirred overnight (17 h). Thebrown reaction mixture was quenched with ice water and extracted withmethylene chloride three times. The combined extracts were dried(MgSO₄), filtered, and concentrated. The residue was purified by flashchromatography on silica gel (0-10% B over 25 min. Bottle A=hexanes,Bottle B=EtOAc) to give 404 mg (53%) of desired product as light brownoil. LC-MS calculated for C₁₆H₁₉F₃N₂O₂: (M+H) 329; found 273.1(M-tBu+1).

Step A-4

4-(Trifluoromethyl)-1′,2′,3′,6′-tetrahydro-2,4′-bipyridine

tert-Butyl4-(trifluoromethyl)-3′,6′-dihydro-2,4′-bipyridine-1′(2′H)-carboxylate(380.0 mg, 1.157 mmol) was dissolved in a 4 M solution of HCl in1,4-dioxane (12.0 mL) to form a light yellow clear (then cloudy)solution. After being stirred at room temperature for 1 h, the reactionmixture was concentrated in vacuo to afford 389 mg of product as ayellow gum. LC-MS calculated for C₁₁H₁₁F₃N₂: (M+H) 229; found 229.1.

Step B

tert-Butyl((1R,3S)-3-isopropyl-3-{[4-(trifluoromethyl)-3′,6′-dihydro-2,4′-bipyridin-1′(2′H)-yl]carbonyl}cyclopentyl)carbamate

To a solution of(1S,3R)-3-[(tert-butoxycarbonyl)amino]-1-isopropylcyclopentanecarboxylicacid (0.288 g, 1.06 mmol) and4-(trifluoromethyl)-1′,2′,3′,6′-tetrahydro-2,4′-bipyridinedihydrochloride (0.320 g, 1.06 mmol) in dry methylene chloride (11.5 mL)was added triethylamine (0.592 mL, 4.25 mmol) followed bybenzotriazol-1-yloxytris(dimethylamino)phospsphonium hexafluorophosphate(0.517 g, 1.17 mmol). After being stirred at room temperature overnight,the brown reaction mixture was washed with NaHCO₃ and brine, dried(MgSO₄), filtered, and concentrated The residue was purified by columnchromatography on silica gel (30:70 EtOAc/hexanes) to provide a lightyellow solid product: 265 mg (52%). LC-MS calculated for C₂₅H₃₄F₃N₃O₃:(M+H) 482; found 382.2 (M-Boc+1).

Step C

(1R,3S)-3-Isopropyl-3-{[4(trifluoromethyl)-3′,6′-dihydro-2,4′-bipyridin-1′(2′H)-yl]carbonyl}cyclopentanamine

tert-Butyl((1R,3S)-3-isopropyl-3-{[4-(trifluoromethyl)-3′,6′-dihydro-2,4′-bipyridin-1′(2′H)-yl]carbonyl}cyclopentyl)carbamate(260.0 mg, 0.54 mmol) was dissolved in a 4 M solution of HCl in1,4-dioxane (6 mL) to form a light yellow clear solution. After beingstirred at room temperature for 1 h, the reaction mixture wasconcentrated in vacuo to afford 300 mg of product as di-HCl salt. Thesolid was treated with a 1 M solution of NaOH. The free base wasextracted with methylene chloride three times. The combined extractswere dried, filtered and concentrated to provide 194 mg (94%) of productas a light yellow gum. LC-MS calculated for C₂₀H₂₆F₃N₃O (M+H) 382; found382.1.

Step D

N-((1R,3S)-3-Isopropyl-3-{[4-(trifluoromethyl)-3′,6′-dihydro-2,4′-bipyridin-1′(2′H)-yl]carbonyl}cyclopentyl)-3-methoxytetrahydro-2H-pyran-4-amine

To a solution of(1R,3S)-3-isopropyl-3-{[4-(trifluoromethyl)-3′,6′-dihydro-2,4′-bipyridin-1′(2′H)-yl]carbonyl}cyclopentanamine(51.2 mg, 0.134 mmol), 3-methoxytetrahydro-4H-pyran-4-one (70 mg, 0.40mmol) and triethylamine (0.0374 mL, 0.269 mmol) in dry methylenechloride (5.0 mL, 0.078 mol) was treated with sodiumtriacetoxyborohydride (85.4 mg, 0.403 mmol) at room temperature under N₂overnight. The reaction was quenched with aqueous NaHCO₃ and dilutedwith methylene chloride. The organic layer was separated and the aqueouslayer was extracted with methylene chloride three times. The combinedextracts were dried over MgSO₄, filtered and evaporated under reducedpressure. The crude product (90 mg) was passed through a short silicagel pad (30:70 MeOH/EtOAc). The filtrate was concentrated and separatedby chiral HPLC to give two isomers: first isomer 26.3 mg; second isomer:17.3 mg. LC-MS calculated for C₂₆H₃₆F₃N₃O₃: (M+H) 496; found 496.2 forboth isomers.

Example 14 Preparation ofN-((1R,3S)-3-isopropyl-3-{[5-(trifluoromethyl)-3′,6′-dihydro-3,4′-bipyridin-1′(2′H)-yl]carbonyl}cyclopentyl)-3-methoxytetrahydro-2H-pyran-4-amine

Step A-1

3-Nitro-5-(trifluoromethyl)pyridin-2-ol

5-(Trifluoromethyl)pyridin-2-ol (10.0 g, 61.31 mmol) was added to astirring concentrated sulfuric acid (50.0 mL) at room temperature. Theresulting clear solution was placed in an ice water bath, and potassiumnitrate (12.4 g, 123 mmol) was added slowly while maintaining thetemperature at 0° C. The resulting mixture was heated at 65° C. for 4 hbefore pouring onto ice and treated carefully with 50% NaOH (83 mL)until pH=8. The aqueous solution was extracted with EtOAc three times.The combined extracts were dried, filtered and concentrated to give 9.78(77%) of crude product (>90% purity) as a yellow solid. Furtherpurification by trituration with EtOAc gave 8.40 g of pure product.LC-MS calculated for C₆H₃F₃N₂O₃: (M+H) 209; found 209.0.

Step A-2

2-Chloro-3-nitro-5-(trifluoromethyl)pyridine

To a solution of phosphoryl chloride (2.0 mL, 21.2 mmol) and quinoline(1.30 mL, 10.8 mmol) was added solid powder3-nitro-5-(trifluoromethyl)pyridin-2-ol (4.00 g, 18.3 mmol) (95%purity). The resulting dark brown thick suspension was heated to refluxfor 4 h and gradually turned into a very cloudy dark brown solution.After cooling to 100° C., water (11 mL) was slowly added to the mixturewhich was further cooled to room temperature and neutralized carefullywith Na₂CO₃. The resulting solution was extracted with EtOAc threetimes. The extracts were combined, dried over MgSO₄, filtered, andevaporated in vacuo. The residue was purified by flash chromatography onsilica gel (EtOAc/hexanes 30:70) to afford 2.28 g of desired product.

Step A-3

5-(Trifluoromethyl)pyridin-3-amine

To a solution of 2-chloro-3-nitro-5-(trifluoromethyl)pyridine (1.25 g,5.518 mmol) in methanol (25.0 mL) under N₂ was added palladium (1.17 g,1.10 mmol) (10% dry weight on wet activated carbon). The reactionmixture was placed on a Parr apparatus and hydrogenated at 50 psi for 90min. The catalyst was filtered off through a celite pad. The filtratewas concentrated to give a crude product (1.08 g) which was pure (>98%by HPLC) enough without further purification. LC-MS calculated forC₆H₅F₃N₂: (M+H) 163; found 163.1.

Step A-4

3-Bromo-5-(trifluoromethyl)pyridine

A solution of sodium nitrite (402 mg, 5.83 mmol) in water (6.8 mL) wasadded slowly to a suspension of 5-(trifluoromethyl)pyridin-3-amine (947mg, 5.55 mmol) in hydrogen bromide (48% aqueous solution, 1.57 mL) inice-water bath. After being stirred for 10 min, the resulting orangediazo solution was directly but slowly transferred to a stirring mixtureof copper(I) bromide (876 mg, 6.11 mmol) and hydrogen bromide (48%aqueous solution, 0.38 mL). The resulting brown mixture was heated at60° C. for 1 h. After cooling to room temperature, the mixture wasdiluted with methylene chloride, washed with 50% NaOH (until pH=11), andwater. The aqueous layer was back extracted with methylene chloride. Thecombined organic extracts were carefully concentrated under vacuum toprovide crude product without purification.

Step A-5

tert-Butyl4-hydroxy-4-[5-(trifluoromethyl)pyridin-3-yl]piperidine-1-carboxylate

To a slightly cloudy solution of 3-bromo-5-(trifluoromethyl)pyridine(2.20 g, 30% purity, 2.92 mmol) in dry methylene chloride (15.0 mL) at−78° C. was added a 1.6 M solution of n-butyllithium in hexanes (1.99mL). After being stirred for 30 min at −78° C., a solution of tert-butyl4-oxo-1-piperidinecarboxylate (0.534 g, 2.65 mmol) in dry methylenechloride (3.0 mL) was added dropwise. The reaction was stirred at −78°C. for 1.5 h and quenched with aqueous NH₄Cl. The resulting solution wasextracted with methylene chloride three times. The combined organiclayers were dried, filtered and concentrated. The residue was purifiedby column chromatography on silica gel (50:50 EtOAc/hexanes) to give 330mg (25%) of desired product as a yellow oil. LC-MS calculated forC₁₆H₂₁F₃N₂O₃: (M+H) 347; found 247.1 (M-Boc+1).

Step A-6

tert-Butyl5-(trifluoromethyl)-3′,6′-dihydro-3,4′-bipyridine-1′(2′H)-carboxylate

To a solution of tert-butyl4-hydroxy-4-[5-(trifluoromethyl)pyridin-3-yl]piperidine-1-carboxylate(300 mg, 0.433 mmol) in pyridine (3.00 mL) cooled in an ice bath wasadded thionyl chloride (0.158 mL, 2.16 mmol). After being stirred atroom temperature overnight (16 h), the brown reaction mixture wasquenched with ice water and extracted with methylene chloride threetimes. The combined extracts were dried (MgSO₄), filtered, andconcentrated. Purification by flash chromatography on silica gel (0-20%B over 35 min. Bottle A=hexanes, Bottle B=EtOAc) provided 65 mg (46%) ofdesired product as a light yellow oil. LC-MS calculated forC₁₆H₁₉F₃N₂O₂: (M+H) 329; found 329.1.

Step A-7

5-(Trifluoromethyl)-1′,2′,3′, 6′-tetrahydro-3,4′-bipyridine

tert-Butyl5-(trifluoromethyl)-3′,6′-dihydro-3,4′-bipyridine-1′(2′H)-carboxylate(50.0 mg, 0.152 mmol) was dissolved in a 4 M solution of HCl in1,4-dioxane (2 mL) to form a light yellow clear (then cloudy) solution.After being stirred at room temperature for 1 h, the reaction mixturewas concentrated in vacuo to afford 40.0 mg (87%) of product as a yellowgum. LC-MS calculated for C₁₁H₁₁F₃N₂: (M+H) 229; found 229.0.

Step B

tert-Butyl((1R,3S)-3Iisopropyl-3-{[5-(trifluoromethyl)-3′,6′-dihydro-3,4′-bipyridin-1′(2′H)-yl]carbonyl}cyclopentyl)carbamate

To a solution of(1S,3R)-3-[(tert-butoxycarbonyl)amino]-1-isopropylcyclopentanecarboxylicacid (40.0 mg, 0.147 mmol) and5-(trifluoromethyl)-1′,2′,3′,6′-tetrahydro-3,4′-bipyridinedihydrochloride (44.4 mg, 0.147 mmol) in dry methylene chloride (2.5 mL)was added triethylamine (0.103 mL, 0.737 mmol) followed bybenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(71.7 mg, 0.162 mmol). After being stirred at room temperatureovernight, the reaction was quenched with aqueous NaHCO₃. The resultingsolution was extracted with methylene chloride three times. The combinedextracts were dried, filtered, concentrated. The residue was purified bysilica gel column (30:70 EtOAc/hexanes, then gradient elution up to50:50 EtOAc/hexanes) to provide a pale yellow gel product: 24 mg (34%).LC-MS calculated for C₂₅H₃₄F₃N₃O₃: (M+H) 482; found 382.0 (M-Boc+1).

Step C

(1R,3S)-3-Isopropyl-3-{[5-(trifluoromethyl)-3′,6′-dihydro-3,4′-bipyridin-1′(2′H)-yl]carbonyl}cyclopentanamine

tert-Butyl((1R,3S)-3-isopropyl-3-{[5-(trifluoromethyl)-3′,6′-dihydro-3,4′-bipyridin-1′(2′H)-yl]carbonyl}cyclopentyl)carbamate(24.0 mg, 0.0498 mmol) was dissolved in a 4 M solution of HCl in1,4-dioxane (2.0 mL) to form a light yellow clear solution. After beingstirred at room temperature for 1 h, the reaction mixture wasconcentrated in vacuo. The residue was treated with a 1 M solution ofNaOH and the solution was extracted with methylene chloride three times.The extracts were dried, filtered and concentrated to provide 28 mg ofproduct as a light yellow solid. LC-MS calculated for C₂₀H₂₆F₃N₃O: (M+H)382; found 382.1.

Step D

N-((1R,3S)-3-Isopropyl-3-{[5-(trifluoromethyl)-3′,6′-dihydro-3,4′-bipyridin-1′(2′H)-yl]carbonyl}cyclopentyl)-3-methoxytetrahydro-2H-pyran-4-amine

To a solution of(1R,3S)-3-isopropyl-3-{[5-(trifluoromethyl)-3′,6′-dihydro-3,4′-bipyridin-1′(2′H)-yl]carbonyl}cyclopentanamine(9.0 mg, 0.024 mmol), 3-methoxytetrahydro-4H-pyran-4-one (12.3 mg,0.0709 mmol) and triethylamine (6.58 μL, 0.0472 mmol) in dry methylenechloride (2.0 mL) was added sodium triacetoxyborohydride (15.0 mg,0.0708 mmol). After being stirred at room temperature overnight, thereaction was quenched with aqueous NaHCO₃ and diluted with methylenechloride. The organic layer was separated and the aqueous layer wasextracted with methylene chloride three times. The organic layers werecombined, dried over MgSO₄, filtered and evaporated under reducedpressure. The crude product (45 mg) was purified by silica gel column(30:70 MeOH/EtOAc) to provide 1.6 mg (14%) of pure product. LC-MScalculated for C₂₆H₃₆F₃N₃O₃: (M+H) 496; found 496.1.

Example 15 Preparation ofN-[(1R,3S)-3-isopropyl-3-({4-[4-(trifluoromethyl)pyrimidin-2-yl]piperazin-1-yl}carbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine

Step A

2-piperazin-1-yl-4-(trifluoromethyl)pyrimidine

A solution of 2-chloro-4-(trifluoromethyl)pyrimidine (2.0 g, 11 mmol),piperazine (2.8 g, 33 mmol) and triethylamine (3.0 mL, 22 mmol) in DMF(10 mL) was stirred at 100° C. in a sealed tube overnight. After removalof most of the solvent, the residue was purified by columnchromatography on silica gel (EtOAc to EtOAc/MeOH/NEt₃ 9/1/0.5) to give1.48 g (56%) of desired product. MS calculated for C₉H₁₁F₃N₄: (M+H) 233;found 233.1.

Step B

tert-Butyl[(1R,3S)-3-isopropyl-3-({4-[4-(trifluoromethyl)pyrimidin-2-yl]piperazin-1-yl}carbonyl)cyclopentyl]carbamate

To a solution of 2-piperazin-1-yl-4-(trifluoromethyl)pyrimidine (250 mg,1.08 mmol),(1S,3R)-3-[(tert-butoxycarbonyl)amino]-1-isopropylcyclopentanecarboxylicacid (300 mg, 1.1 mmol) and triethylamine (0.45 mL, 3.2 mmol) inmethylene chloride (10 mL) was addedbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(520 mg, 1.2 mmol). After being stirred overnight, the reaction wasquenched with saturated NaHCO₃. The resulting solution was extractedwith EtOAc three times. The combined organic layers were dried (MgSO₄)and concentrated. Purification by column chromatography on silica gel(20%-40% EtOAc/hexanes) provided 290 mg of desired product MS calculatedfor C₂₃H₃₄F₃N₅O₃: (M+H) 486; found 386.1 (M-Boc+1).

Step C

(1R,3S)-3-Isopropyl-3-({4-[4-(trifluoromethyl)pyrimidin-2-yl]piperazin-1-yl}carbonyl)cyclopentanamine

tert-Butyl[(1R,3S)-3-isopropyl-3-({4-[4-(trifluoromethyl)pyrimidin-2-yl]piperazin-1-yl}carbonyl)cyclopentyl]carbamate(290 mg, 0.60 mmol) was dissolved in a 4.0 M solution of HCl in1,4-dioxane (10 mL). After being stirred at room temperature for 1 h,the mixture was concentrated to give 270 mg of desired product. MScalculated for C₁₈H₂₆F₃N₅O: (M+H) 386; found 386.1.

Step D

N-[(1R,3S)-3-Isopropyl-3-({4-[4-(trifluoromethyl)pyrimidin-2-yl]piperazin-1-yl}carbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine

To a solution of(1R,3S)-3-isopropyl-3-({4-[4-(trifluoromethyl)pyrimidin-2-yl]piperazin-1-yl}carbonyl)cyclopentanaminedihydrochloride (135.0 mg, 0.2945 mmol),3-methoxytetrahydro-4H-pyran-4-one (110 mg, 0.88 mmol) and triethylamine(0.16 mL, 1.2 mmol) in methylene chloride (8 mL) was added sodiumtriacetoxyborohydride (190 mg, 0.88 mmol). After being stirred at roomtemperature overnight, the reaction was quenched with saturated NaHCO₃.The resulting solution was extracted with EtOAc three times. Thecombined organic layers were dried (MgSO₄), concentrated. The residuewas purified by flash chromatography on silica gel (EtOAc toEtOAc/Et₃N=10:0.1) to give 123 mg of desired product as a mixture of twoisomers. The two isomers were separated by chiral HPLC to give isomer 1(65 mg after converting to TFA salt) and isomer 2 (45 mg afterconverting to TFA salt). LCMS calculated for C₂₄H₃₆F₃N₅O₃: (M+1) 500;found 500.1 for both isomers.

Example 16 Preparation ofN-[(1R,3S)-3-isopropyl-3-({4-[6-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}carbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine

Step A

1-[6-(Trifluoromethyl)pyridin-2-yl]piperazine

A solution of 2-chloro-6-(trifluoromethyl)pyridine (1.0 g, 5.5 mmol),piperazine (1.4 g, 16.0 mmol), and triethylamine (1.5 mL, 11.0 mmol)were mixed in DMF (10 mL) in a sealed tube. The mixture was heated at100° C. overnight. The reaction mixture was concentrated andchromatographed on silica gel (ethyl acetate to EA/MeOH/Et₃N=9:1:0.5) togive 1.05 g of the desired product. MS calculated for C₁₀H₁₂F₃N₃: (M+H)232.1; found 232.1.

Step B

tert-Butyl[(1R,3S)-3Iisopropyl-3-(4-[6-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate

To a solution of 1-[6-(trifluoromethyl)pyridin-2-yl]piperazine (249 mg,1.08 mmol),(1S,3R)-3-[(tert-butoxycarbonyl)amino]-1-isopropylcyclopentanecarboxylicacid (300 mg, 1.10 mmol), triethylamine (0.45 mL, 3.2 mmol) in methylenechloride (10 mL) was addedbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(524 mg, 1.18 mmol). After being stirred overnight, the reaction wasquenched with saturated sodium NaHCO₃. The resulting solution wasextracted with EtOAc three times. The combined organic layers were dried(MgSO₄), concentrated, and purified by column chromatography on silicagel (20% EA/hex to 40% EA/hexanes) to provide 310 mg of the desiredproduct. MS calculated for C₂₄H₃₆F₃N₄O₃: (M+H) 485.3; found 485.3.

Step C

(1R,3S)-3-Isopropyl-3-(4-[6-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentanaminedihydrochloride

tert-Butyl[(1R,3S)-3-isopropyl-3-(4-[6-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate(300 mg, 0.62 mmol) was dissolved in a 4.0 M solution of HCl in1,4-dioxane (10 mL). After being stirred at room temperature for 1 h,the solution was concentrated to give 260 mg of desired product. MScalculated for C₁₉H₂₇F₃N₄O: (M+H) 385.2; found 385.2.

Step D

N-[(1R,3S)-3-Isopropyl-3-({4-[6-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}carbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine

To a solution of(1R,3S)-3-isopropyl-3-(4-[6-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentanaminedihydrochloride (110 mg, 0.24 mmol), 3-methoxytetrahydro-4H-pyran-4-one(80 mg, 0.61 mmol), and triethylamine (0.16 mL, 1.2 mmol) in methylenechloride (8 mL) was added sodium triacetoxyborohydride (190 mg, 0.88mmol). After being stirred at room temperature overnight, the reactionwas quenched with saturated NaHCO₃. The resulting solution was extractedwith EtOAc three times. The combined organic layers were dried (MgSO₄)and concentrated. The residue was purified by flash chromatography onsilica gel (EtOAc to EtOAc/Et₃N=10:0.1) to give 123 mg of desiredproduct as a mixture of two isomers. The two isomers were separated bychiral HPLC to give isomer 1 (45 mg after converting to TFA salt) andisomer 2 (35 mg after converting to TFA salt). LCMS calculated forC₂₅H₃₇F₃N₄O₃: (M+H) 498.2; found 498.2 for both isomers.

Example 17 Preparation ofN-[(1R,3S)-3-isopropyl-3-(4-[6-(trifluoromethyl)pyrimidin-4-yl]piperazin-1-ylcarbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine

Step A

4-Chloro-6-(trifluoromethyl)pyrimidine

A solution of 6-(trifluoromethyl)pyrimidin-4-ol (5.0 g, 30.5 mmol),phosphoryl chloride (3.41 mL, 36.6 mmol), and quinoline (2.16 mL, 18.3mmol) in toluene (50 mL) was stirred at 100° C. for 5 h. The reactionwas diluted with water and extracted with ethyl acetate three times,dried with sodium sulfate, filtered, and concentrated in vacuo. Thecrude residue was purified by flash column chromatography (10%EtOAc/Hexane) to yield the desired product (1.20 g, 21.6%). ¹H NMR (400MHz, CDCl3): 9.21 ppm (1H, s), 7.78 (1H, s).

Step B

4-piperazin-1-yl-6-(trifluoromethyl)pyrimidine

A solution of 4-chloro-6-(trifluoromethyl)pyrimidine (1.0 g, 5.48 mmol),piperazine (2.36 g, 27.4 mmol), and triethylamine (2.29 mL, 16.4 mmol)in DMF (20 mL) was stirred at 100° C. for 5 h. The reaction solution wasdiluted with water and extracted with ethyl acetate three times, driedwith sodium sulfate, filtered, and concentrated in vacuo. The cruderesidue was purified by flash column chromatography (10% MeOH/5%Et₃N/EtOAc) to yield the desired product (720 mg, 56.6%). LCMScalculated for C₉H₂F₃N₄: (M+H) 233.1; found 233.1.

Step C

tert-Butyl[(1R,3S)-3-Isopropyl-3-(4-[6-trifluoromethyl)pyrimidin-4-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate

A solution of 4-piperazin-1-yl-6-(trifluoromethyl)pyrimidine (1.0 g,4.31 mmol),(1S,3R)-3-[(tert-butoxycarbonyl)amino]-1-isopropylcyclopentanecarboxylicacid (1.75 g, 6.46 mmol),benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(2.86 g, 6.46 mmol), and triethylamine (1.20 mL, 8.61 mmol) in methylenechloride (10 mL) was stirred at room temperature overnight. The reactionmixture was diluted with methylene chloride, washed with brine, driedwith sodium sulfate, filtered, and concentrated in vacuo. The cruderesidue was purified by flash column chromatography to yield the desiredproduct (800 mg, 38.3%). LCMS calculated for C₂₃H₃₅F₃N₅O₃: (M+H) 486.2;found 486.2.

Step D

A solution of tert-butyl[(1R,3S)-3-isopropyl-3-(4-[6-(trifluoromethyl)pyrimidin-4-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate(800 mg, 1.65 mmol) dissolved in 4 M of HCl in 1,4-dioxane (10 ml, 40mmol) was stirred at room temperature for 2 h. The reaction mixture wasdiluted with methylene chloride, washed with saturated NaHCO₃ solution,dried with sodium sulfate, filtered, and concentrated in vacuo. Thecrude residue was purified by flash column chromatography to yield thedesired product (0.6 g, 99%). LCMS calculated for C₁₈H₂₇F₃N₅O: (M+H)386.2; found 386.2.

Step E

N-[(1R,3S)-3-Isopropyl-3-(4-[6-(trifluoromethyl)pyrimidin-4-yl]piperazin-1-ylcarbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine

To a solution of(1R,3S)-3-isopropyl-3-(4-[6-(trifluoromethyl)pyrimidin-4-yl]piperazin-1-ylcarbonyl)cyclopentananine(120 mg, 0.30 mmol), 3-methoxytetrahydro-4H-pyran-4-one (120 mg, 0.90mmol), and triethylamine (0.12 mL, 0.90 mmol) in methylene chloride (20mL) was added sodium triacetoxyborohydride (0.19 g, 0.90 mmol). Afterbeing stirred at room temperature overnight, the reaction mixture wasdiluted with methylene chloride, washed with brine, dried over sodiumsulfate, filtered and concentrated. The residue was purified by flashchromatography to provide the desired product as a mixture of fourisomers. LCMS calculated for C₂₄H₃₇F₃N₅O₃: (M+H) 500.3; found 500.3 forfour isomers.

Example 18 Preparation ofN-[(1R,3S)-3-isopropyl-3-(4-[6-methyl-4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine

Step A

1-[6-Methyl-4-(trifluoromethyl)pyridin-2-yl]piperazine

A solution of 2-chloro-6-methyl-4-(trifluoromethyl)pyridine (1.0 g, 5.11mmol), piperazine (1.32 g, 15.3 mmol), and triethylamine (0.71 mL, 5.1mmol) were mixed in 1,4-dioxane (10 mL). After stirring at 100° C. for 5h, the reaction solution was diluted with water and extracted with ethylacetate three times, dried with sodium sulfate, filtered, andconcentrated in vacuo. The crude residue was purified by flash columnchromatography (10% MeOH/5% Et₃N/EtOAc) to yield the desired product(880 mg, 70.2%). LCMS calculated for C₁₁H₁₅F₃N₃: (M+H) 246.1; found246.1.

Step B

tert-Butyl[(1R,3S)-3-isopropyl-3-(4-[6-methyl-4-(trifluormethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate

To a solution of 1-[6-methyl-4-(trifluoromethyl)pyridin-2-yl]piperazine(280 mg, 1.1 mmol),(1S,3R)-3-[(tert-butoxycarbonyl)amino]-1-isopropylcyclopentanecarboxylicacid (460 mg, 1.7 mmol) in methylene chloride (30 mL) was addedbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(0.60 g, 1.4 mmol), and triethylamine (0.20 g, 2.0 mmol). After beingstirred overnight, the reaction mixture was diluted with methylenechloride, washed with brine, dried over sodium sulfate, filtered andconcentrated. The crude residue was purified by flash columnchromatography to provide the desired product (200 mg, 35.1%). LCMScalculated for C₂₅H₃₇F₃N₄O₃: (M+H) 499.3; found 499.2.

Step C

(1R,3S)-3-Isopropyl-3-(4-[6-methyl-4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentanamine

A solution of tert-butyl[(1R,3S)-3-isopropyl-3-(4-[6-methyl-4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate(200 mg, 1.65 mmol) dissolved in 4 M of HCl in 1,4-dioxane (10 mL, 40mmol) was stirred at room temperature for 1 h. The reaction was dilutedwith methylene chloride, washed with saturated NaHCO₃ solution, driedwith sodium sulfate, filtered, and concentrated in vacuo to provide thedesired product (0.15 g, 94%). LCMS calculated for C₂₀H₃₀F₃N₄O: (M+H)399.2; found 399.2.

Step D

N-[(1R,3S)-3-Isopropyl-3-(4-[6-methyl-4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine

To a solution of(1R,3S)-3-isopropyl-3-(4-[6-methyl-4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentanamine(120 mg, 0.30 mmol), 3-methoxytetrahydro-4H-pyran-4-one (120 mg, 0.90mmol), and triethylamine (0.12 mL, 0.90 mmol) in methylene chloride (20mL) was added sodium triacetoxyborohydride (0.19 g, 0.90 mmol). Afterbeing stirred at room temperature overnight, the reaction mixture wasdiluted with methylene chloride, washed with brine, dried over sodiumsulfate, filtered and concentrated. The residue was purified by flashchromatography to provide the desired product as a mixture of cis/transisomers. LCMS calculated for C₂₆H₄₀F₃N₄O₃: (M+H) 513.3; found 513.2 forboth isomers.

Example 19 Preparation of(4R)—N-[(1R,3S)-3-isopropyl-3-(4-[3-(trifluoromethyl)phenyl]piperidin-1-ylcarbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine

(4R)-N-((1R,3S)-3-Isopropyl-3-[4-[3-(trifluoromethyl)phenyl]-3,6-dihydropyridin-1(2H)-yl]carbonylcyclopentyl)-3-methoxytetrahydro-2H-pyran-4-amine(8.0 mg, 0.016 mmol) was dissolved in methanol (0.63 mL),degassed-purged with N₂, followed by the addition of Palladium (3.44 mg)(10% dry weight on wet activated carbon). The reaction flask wasdegassed-purged with N₂ three times, then stirred at room temperatureunder H₂ (1 atm) overnight. The mixture was filtered through celite pad,washed with methylene chloride and concentrated to provide the desiredproduct as a white solid (5.7 mg, 71%). LC-MS calculated forC₂₇H₄₀F₃N₂O₃: (M+H) 497.2; found 497.2.

Example 20 Preparation of2-[(1R,3S)-3-[(3-methoxytetrahydro-2H-pyran-4-yl)amino]-1-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]propan-2-olbis(trifluoroacetate) (salt)

Step A

Methyl(1R,4S)-4-[(tert-Butoxycarbonyl)amino]-1-(1-hydroxy-1-methylethyl)cyclopent-2-ene-1-carboxylate

To a 1.00 M solution of lithium hexamethyldisilazide in THF (45 mL)stirring at −78° C. was added a solution of methyl(1R,4S)-4-[(tert-butoxycarbonyl)amino]cyclopent-2-ene-1-carboxylate (5.0g, 21 mmol) in THF (40 mL). The resulting golden mixture was warmed to−28° C. to −23° C. (CCl₄/dry ice) and stirred for 30 min. The reactionsolution was cooled down to −78° C. and anhydrous acetone (1.8 mL, 25mmol) was added. After the addition, the reaction mixture was kept in aCCl₄/dry ice bath and allowed to warm to room temperature overnight. Thedark solution was quenched with saturated NH₄Cl and extracted with etherthree times. The combined extracts were dried, filtered, concentrated.The crude residue was purified by flash column chromatography(EtOAc/Hexane) to yield the desired product (1.4 g, 22.6%).

Step B

Methyl(3S)-3-[(tert-Butoxycarbonyl)amino]-1-(t-hydroxy-1-methylethyl)cyclopentanecarboxylate

Methyl(4S)-4-[(tert-butoxycarbonyl)amino]-1-(1-hydroxy-1-methylethyl)cyclopent-2-ene-1-carboxylate(1.4 g, 4.7 mmol) was dissolved in ethanol (30 mL) in a Parr flask andpurged with N₂. 10% Palladium on carbon (0.14 g) was added and themixture was shaken overnight under 50 psi Hz. The mixture was filteredthrough celite, washed with methylene chloride and concentrated to givethe desired product (1.06 g, 86%).

Step C

(3S)-3-[(tert-Butoxycarbonyl)amino]-1-(1-hydroxy-1-methylethyl)cyclopentanecarboxylicacid

To a solution of methyl(3S)-3-[(tert-butoxycarbonyl)amino]-1-(1-hydroxy-1-methylethyl)cyclopentanecarboxylate(1.0 g, 3.3 mmol) in a mixture of THF (30 mL), methanol (30 mL) andwater (6 mL) was added lithium hydroxide monohydrate (0.22 g, 5.3 mmol)and the mixture refluxed overnight (110° C.). The organic solvents wereevaporated and the aqueous layer was washed with ether one time. Theaqueous layer was then acidified with 6N HCl to about pH 4 and extractedwith methylene chloride three times. The combined extracts were dried,filtered, and concentrated to provide the desired product (0.47 g, 49%).

Step D

tert-Butyl[3-(1-Hydroxy-1-methylethyl)-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate

To a suspension of(3S)-3-[(tert-butoxycarbonyl)amino]-1-(1-hydroxy-1-methylethyl)cyclopentanecarboxylicacid (150 mg, 0.52 mmol) and1-[4-(trifluoromethyl)pyridin-2-yl]piperazine (130 mg, 0.57 mmol) inmethylene chloride (3 mL) under N₂ was added triethylamine (0.16 g, 1.6mol) and benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (0.25 g, 0.57 mmol). After being stirred at roomtemperature overnight, the reaction was quenched by saturated NaHCO₃solution and extracted with methylene chloride three times. The combinedextracts were dried (MgSO₄), filtered, concentrated and purified byflash column chromatography to yield the desired product (76 mg, 29%).

Step E

2-[(1R,3S)-3-Amino-1-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]propan-2-olDihydrochloride

tert-Butyl[(3R)-3-(1-hydroxy-1-methylethyl)-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate(75 mg, 0.15 mmol) was mixed with a 2.00 M solution of hydrogen chloridein ether (2 mL) and THF (1 mL). After being stirred for 1 h at roomtemperature, the reaction solution was concentrated to provide thedesired product (70 mg, 98.7%). LCMS calculated for C₁₇H₂₈F₃N₄O₂: (M+H)473.2; found 473.2.

Step F

2-[(1R,3S)-3-[(3-Methoxytetrahydro-2H-pyran-4-yl)amino]-1-(4-[4-trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]propan-2-olBis(trifluoroacetate)

To a solution of2-[(1R,3S)-3-amino-1-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]propan-2-oldihydrochloride (71 mg, 0.15 mmol), 3-methoxytetrahydro-4H-pyran-4-one(69 mg, 0.45 mmol), and triethylamine (63 μL, 0.45 mmol) in methylenechloride (2 mL) was added sodium triacetoxyborohydride (64 mg, 0.30mmol). After being stirred at room temperature overnight, the reactionwas quenched with saturated NaHCO₃ and extracted with methylene chloridethree times. The combined extracts were dried (MgSO₄), filtered,concentrated, purified by chromatography and then converted to thedesired product TFA salts (57 mg, 57.5%). LCMS calculated forC₂₅H₃₇F₃N₄O₄: (M+H) 515.3; found 515.4.

Example 21 Preparation of2-[(1S,3R)-3-[(4R)-3-methoxytetrahydro-2H-pyran-4-yl]amino-1-(4-[4-(trifluoromethyl)pyrimidin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]propan-2-olbis(trifluoroacetate)

The title compound was prepared using procedures analogous to thosedescribed for Example 20. MS calculated for C₂₄H₃₆F₃N₅O₄: (M+H) 516.3;found 516.4.

Example 22 Preparation of2-[(1S,3S)-3-[(3-methoxytetrahydro-2H-pyran-4-yl)amino]-1-(4-[6-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]propan-2-olbis(trifluoroacetate)

The title compound was prepared using procedures analogous to thosedescribed for Example 20. MS calculated for C₂₅H₃₇F₃N₄O₄: (M+H) 515.3;found 515.4.

Example 23 Preparation ofN-[(1S,3S)-3-ethyl-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-aminebis(trifluoroacetate)

Step A

Methyl(1R,4S)-4-[(tert-Butoxycarbonyl)amino]-1-ethylcyclopent-2-ene-1-carboxylate

To a 1.00 M solution of lithium hexamethyldisilazide in THF (61.5 mL,61.5 mmol) was added a solution of methyl(1R,4S)-4-[(tert-butoxycarbonyl)amino]cyclopent-2-ene-1-carboxylate(6.71 g, 27.8 mmol) in THF (10.0 mL) at −78° C. over 10 min. Theresulted light brown solution was stirred at −78° C. for 30 min beforeiodoethane (2.67 mL, 33.4 mmol) was added in one portion. The mixturewas then kept at −25° C. overnight. The reaction was quenched withaqueous NH₄Cl. The organic layer was separated and the aqueous layer wasfurther extracted with ether three times. The combined organic layerswere then washed with brine, dried over Na₂SO₄, filtered, concentratedand purified by flash column chromatography to yield the desired productas a 7:1 cis/trans mixture (4.83 g, 65%). MS calculated for C₁₄H₂₃NO₄:(M+H) 170.2; found 170.1 (M+H-Boc).

Step B

(1R,4S)-4-[(tert-Butoxycarbonyl)amino]-1-ethylcyclopent-2-ene-1-carboxylicAcid

To a solution of methyl(1R,4S)-4-[(tert-butoxycarbonyl)amino]-1-ethylcyclopent-2-ene-1-carboxylate(4.80 g, 17.8 mmol)) in a mixture of tetrahydrofuran (100 mL), methanol(100 mL) and water (20 mL) was added lithium hydroxide, monohydrate (1.2g, 28.6 mmol) and the mixture was refluxed overnight. The organicsolvents were evaporated. The aqueous layer was then acidified with 6NHCl to about pH 4 and extracted with methylene chloride three times. Thecombined extracts were dried, filtered, and concentrated to give amixture of cis/trans isomers (2.93 g, cis/trans=7:1) as a light yellowsolid. This solid was dissolved in EtOAc (4.0 mL) with heating anddiluted with hexanes (100 mL) to give a clear solution. This solutionwas allowed to cool to room temperature slowly over 1 h and thenmaintained at −25° overnight. The cis-isomer was crystallized and driedto provide the desired product (1.40 g, 31%) as a white solid. MScalculated for C₁₃H₂₁NO₄: (M+H) 256.2.2; found 156.1 (M+H-Boc).

Step C

(1S,3R)-3-[(tert-Butoxycarbonyl)amino]-1-ethylcyclopentanecarboxylicAcid

To a solution of(1R,4S)-4-[(tert-butoxycarbonyl)amino]-1-ethylcyclopent-2-ene-1-carboxylicacid (1.38 g, 5.41 mmol) in ethanol (40 mL) was added 10% palladium oncarbon (200 mg). The mixture was shaken under hydrogen at 50 psi for 18h and filtered through celite. The filtrate was evaporated in vacuo toafford the desired product (1.5 g). MS calculated for C₁₃H₂₃NO₄: (M+H)258.2; found 158.1 (M+H-Boc).

Step D

tert-Butyl[(1S,3R)-3-Ethyl-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate

To a solution of(1R,3S)-3-[(tert-butoxycarbonyl)amino]-1-ethylcyclopentanecarboxylicacid (0.30 g, 1.2 mmol) and1-[4-(trifluoromethyl)pyridin-2-yl]piperazine dihydrochloride (0.39 g,1.3 mmol) in DMF (10 mL) under N2 was added triethylamine (0.65 mL, 4.7mmol) and O-(Benzotriazol-1-yl)-N,N,N′,N-tetramethyluroniumhexafluorophosphate (0.663 g, 1.75 mmol). After being stirred at roomtemperature overnight, the reaction was quenched with saturated NaHCO₃and extracted with methylene chloride three times. The combined extractswere dried (MgSO₄), filtered, concentrated and purified by flash columnchromatography to yield the desired product (400 mg, 72.9%). MScalculated for C₂₃H₃₄F₃N₄O₃: (M+H) 471.3; found 371.2 (M+H-Boc).

Step E

(1S,3R)-3-Ethyl-3-(4-[4-(Trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentanaminedihydrochloride

tert-Butyl[(1S,3R)-3-ethyl-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate(0.39 g, 0.83 mmol) was dissolved in a 4M solution of hydrogen chloridein 1,4-dioxane (3.1 mL) and the solution was stirred at room temperatureovernight. The reaction solution was evaporated to give the desiredproduct as a yellow powder (0.36 g, 96%).

Step F

N-[(1S,3S)-3-Ethyl-3-(4-[4-(Trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amineBis(trifluoroacetate)

To a solution of(1S,3S)-3-ethyl-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentanaminedihydrochloride (100 mg, 0.20 mmol), 3-methoxytetrahydro-4H-pyran-4-one(77 mg, 0.50 mmol), and triethylamine (0.110 mL, 0.79 mmol) in methylenechloride (3 mL) was added sodium triacetoxyborohydride (96 mg, 0.45mmol). After being stirred at room temperature overnight, the reactionwas quenched with saturated NaHCO₃ and extracted with methylene chloridethree times. The combined organics were dried (MgSO₄), filtered,concentrated, purified by flash column chromatography (NH₄OH/MeOH/EtOAc)and converted to salt to yield the desired product (111 mg, 69.1%). MScalculated for C₂₄H₃₅F₃N₄O₃: (M+H) 485.3; found 485.2.

Example 24 Preparation of(4R)-N-[(1R,3S)-3-ethyl-3-(4-[4-(trifluoromethyl)pyrimidin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-aminebis(trifluoroacetate)

The title compound was prepared using procedures analogous to thosedescribed for Example 23. MS calculated for C₂₃H₃₄F₃N₅O₃: (M+H) 486.3;found 486.2.

Example 25 Preparation ofN-[(1S,3S)-3-ethyl-3-(4-[6-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-aminebis(trifluoroacetate)

The title compound was prepared using procedures analogous to thosedescribed for Example 23. MS calculated for C₂₄H₃₅F₃N₄O₃: (M+H) 485.3;found 485.3.

Example 26 Preparation of(4R)-N-[(1R,3S)-3-methyl-3-(4-[4-(trifluoromethyl)pyrimidin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-aminebis(trifluoroacetate)

The title compound was prepared using procedures analogous to thosedescribed for Example 23. MS calculated for C₂₂H₃₂F₃N₅O₃: (M+H) 472.3.3;found 472.3.

Example 27 Preparation of(4R)-3-methoxy-N-[(1R,3S)-3-(2-methoxyethyl)-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]tetrahydro-2H-pyran-4-aminebis(trifluoroacetate)

Step A I-Iodo-2-methoxyethane

To a solution of 1-bromo-2-methoxyethane (2.0 g, 14 mmol) in acetone (40mL) was added sodium iodide (11 g, 72 mmol) and the resulting solutionwas refluxed (70° C.) under N₂ for 3 hours. The mixture was cooled andfiltered. Upon further cooling in the refrigerator, additional solidscrashed out and were filtered off before concentrating to give an orangeresidue. The residue was taken up in ether and washed with Na₂S₂O₃,which rendered a nearly colorless solution. The solution was dried(MgSO₄), filtered, and concentrated to give a yellow oil (1.8 g, 64%).¹H NMR (CDCl₃) δ 3.70-3.60 (2H, t, J=5 Hz), 3.40 (3H, s), 3.30-3.20 (2H,t, J=5 Hz).

Step B

Methyl(1S,4S)-4-[(tert-Butoxycarbonyl)amino]-1-(2-methoxyethyl)cyclopent-2-ene-1-carboxylate

To a 1.00 M solution of lithium hexamethyldisilazide in tetrahydrofuran(9.1 mL, 9.1 mmol) under N₂ at −78° C. was added a solution of methyl(1R,4S)-4-[(tert-butoxycarbonyl)amino]cyclopent-2-ene-1-carboxylate (1.0g, 4.1 mmol) in tetrahydrofuran (2.0 mL). The resulted light brownsolution was stirred at −78° C. for 30 min before adding a solution of1-iodo-2-methoxyethane (0.93 g, 5.0 mmol) in tetrahydrofuran (2.0 mL).The mixture was stirred for an hour at −78° C. then kept in a freezerreading at −20° C. overnight. The reaction was quenched with saturatedammonium chloride. The layers were separated and the aqueous extractedwith ether three times. The combined organics were then washed withbrine, dried (MgSO₄), filtered and purified by flash chromatography(EtOAc/Hexane) to provide the desired product (0.28 g, 23%). LCMScalculated for C₅H₂₆NO₅: (M+H) 300.2; found 300.2.

Step C

(1S,4S)-4-[(tert-Butoxycarbonyl)amino]-1-(2-methoxyethyl)cyclopent-2-ene-1-carboxylicacid

To a stirred solution of methyl(1S,4S)-4-[(tert-butoxycarbonyl)amino]-1-(2-methoxyethyl)cyclopent-2-ene-1-carboxylate(0.78 g, 2.6 mmol) in tetrahydrofuran (1.5 mL), methanol (15 mL), andwater (3.0 mL) was added lithium hydroxide monohydrate (0.55 g, 13 mmol)and the resulting orange mixture was stirred at 80° C. overnight. Thesolvents were evaporated and the mixture was acidified with 6 N HCl to apH of about 4. The aqueous was then extracted with methylene chloridethree times. The combined organic layers were dried (MgSO₄), filtered,and concentrated in vacuo to give the desired product as an oil (0.47 g,63.2%). LCMS calculated for C₁₄H₂₄NO₅: (M+H) 286.2; found 286.2.

Step D

(1S,3R)-3-[(tert-Butoxycarbonyl)amino]-1-(2-methoxyethyl)cyclopentanecarboxylicacid

To a solution of(1S,4S)-4-[(tert-butoxycarbonyl)amino]-1-(2-methoxyethyl)cyclopent-2-ene-1-carboxylicacid (1.56 g, 5.47 mmol) in methanol (30 mL) was added 10% palladium oncarbon (150 mg). The mixture was shaken under hydrogen at 50 psiovernight and filtered through celite. The filtrate was evaporated invacuo to afford the desired product (1.57 g, 99.9%). MS calculated forC₁₄H₂₆NO₅ (M+H) 288.2; found 188.2 (M+H-Boc).

Step E

tert-Butyl[(1R,3S)-3-(2-Methoxyethyl)-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate

(1S,3R)-3-[(tert-Butoxycarbonyl)amino]-1-(2-methoxyethyl)cyclopentanecarboxylicacid (276.6 mg, 0.96 mmol),1-[4-(trifluoromethyl)pyridin-2-yl]piperazine dihydrochloride (322.0rug, 1.06 mmol), triethylamine (0.54 mL, 3.85 mmol) andO-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(547.6 mg, 1.44 mmol) (HBTU) were mixed in dry DMF (6.6 mL) and theresultanting brown solution was stirred at room temperature under N₂ forthree days. The reaction mixture was diluted with CH₂Cl₂, and washedwith saturated Na₂CO₃. The aqueous layer was extracted with CH₂Cl₂ fourtimes. The combined organic layers were dried (MgSO₄), filtered,concentrated and purified by flash chromatography (EtOAc/Hexane) toprovide the desired product (252 mg, 52%). MS calculated forC₂₄H₃₆F₃N₄O₄ (M+H) 501.3; found 401.3 (M+H-Boc).

Step F

(1R,3S)-3-(2-Methoxyethyl)-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentanamineDihydrochloride

tert-Butyl[(1R,3S)-3-(2-methoxyethyl)-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate(252 mg, 0.503 mmol) was dissolved in a 2 M solution of hydrogenchloride in ether (8 mL) and stirred at room temperature for 2 h. Thereaction solution was concentrated to give the desired product as ayellow powder (0.36 g, 96%). MS calculated for C₁₉H₂₇F₃N₄O₂ (M+H) 401.3;found 401.3.

Step G

(4R)-3-Methoxy-N-[(1R,3S)-3-(2-methoxyethyl)-3-(4-([4-trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]tetrahydro-2H-pyran-4-amineBis(trifluoroacetate)

To a solution of(1R,3S)-3-(2-methoxyethyl)-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentanaminedihydrochloride (140.0 mg, 0.30 mmol),3-methoxytetrahydro-4H-pyran-4-one (115 mg, 0.887 mmol) andtriethylamine (0.165 mL, 1.18 mmol) in dry methylene chloride (12 mL)was added sodium triacetoxyborohydride (188.1 mg, 0.887 mmol). Afterbeing stirred under N₂ at room temperature for three days, the reactionwas quenched with saturated NaHCO₃ and extracted with methylene chloridethree times. The combined organics were dried (MgSO₄), filtered,concentrated, purified by flash column chromatography (MeOH/EtOAc) andconverted to TFA salt to yield the desired product (72.4 mg, 34%). MScalculated for C₂₅H₃₇F₃N₄O₄: (M+H) 515.3; found 515.4.

Example 28 Preparation of3-methoxy-N-[(1S,3S)-3-(2-methoxyethyl)-3-(4-[4-(trifluoromethyl)pyrimidin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]tetrahydro-2H-pyran-4-aminebis(trifluoroacetate)

The title compound was prepared using procedures analogous to thosedescribed for Example 27. MS calculated for C₂₄H₃₆F₃N₅O₄: (M+H) 516.3;found 516.3.

Example 29 Preparation of(4R)-N-[(1R,3S)-3-(ethoxymethyl)-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-aminebis(trifluoroacetate)

Step A

Methyl(1R,4S)-4-[(tert-Butoxycarbonyl)amino]-1-(ethoxymethyl)cyclopent-2-ene-1-carboxylate

To a 1.0 M solution of lithium hexamethyldisilazide in tetrahydrofuran(36.7 mL) was added a solution of methyl(1R,4S)-4-[(tert-butoxycarbonyl)amino]cyclopent-2-ene-1-carboxylate(4.00 g, 1.66 mmol) in tetrahydrofuran (6.0 mL) at −78° C. The resultinglight brown solution was stirred at −78° C. for 30 min before(chloromethoxy)ethane (1.88 g, 19.9 mol) was added in one portion. Themixture was stirred at −78° C. for 1 h and then kept in a freezerreading at −25° C. overnight. The reaction was then quenched withsaturated NH₄Cl (50 mL). The organic layer was separated and the aqueouslayer was extracted with CH₂Cl₂ three times. The combined organic layerswere washed with brine, dried over Na₂SO₄, filtered, concentrated, andpurified by flash chromatography (0 to 15% EtOAC in hexanes) to providethe desired product (3.29 g, 66%) as a cis/trans (3:2) mixture based onthe analysis on reverse phase HPLC. MS calculated for C₁₅H₂₆NO₅: (M+H)300.2; found 200.2 (M+H-Boc).

Step B

(1R,4S)-4-[(tert-Butoxycarbonyl)amino]-1-(ethoxymethyl)cyclopent-2-ene-1-carboxylicAcid

To a solution of methyl(1R,4S)-4-[(tert-butoxycarbonyl)amino]-1-(ethoxymethyl)cyclopent-2-ene-1-carboxylate(3.25 g, 10.8 mmol) in tetrahydrofuran (58.7 mL), methanol (58.7 mL) andwater (12.6 mL) was added lithium hydroxide monohydrate (0.731 g, 17.42mmol). The pink mixture was heated to reflux overnight. The organicsolvents were removed in vacuo and the aqueous layer was washed oncewith ether and then acidified slowly with concentrated HCl until the pHreached 4. The resulting suspension was extracted with methylenechloride three times. The combined organic layers were dried over MgSO₄,filtered and concentrated to give the desired product as a mixture ofcis/trans isomers (2.75 g, 89%). MS calculated for C₁₄H₂₄NO₅: (M+H)286.2; found 186.2 (M+H-Boc).

Step C

(1S,3R)-3-[(tert-Butoxycarbonyl)amino]-1-(ethoxymethyl)cyclopentanecarboxylicAcid

To a solution of(1R,4S)-4-[(tert-butoxycarbonyl)amino]-1-(ethoxymethyl)cyclopent-2-ene-1-carboxylicacid (2.70 g, 9.46 mmol) in ethanol (69.5 mL) was added 10% palladium oncarbon (350 mg). The mixture was shaken under hydrogen at 50 psi for 18h, filtered through celite and washed with methylene chloride. Thefiltrate was concentrated to afford the desired product (2.87 g). MScalculated for C₁₄H₂₆NO₅ (M+H) 288.2; found 188.2 (M+H-Boc).

Step D

tert-Butyl[(3S)-3-(Ethoxymethyl)-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate

(1S)-3-[(tert-Butoxycarbonyl)amino]-1-(ethoxymethyl)cyclopentanecarboxylicacid (429.4 mg, 1.494 mmol),1-[4-(trifluoromethyl)pyridin-2-yl]piperazine dihydrochloride (500.0 mg,1.644 mmol), triethylamine (0.833 mL, 5.98 mmol) andO-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(850.3 mg, 2.242 mmol) (HBTU) were mixed in dry DMF (10.2 mL). Theresulting brown solution was stirred at room temperature under N₂overnight. The reaction mixture was diluted with CH₂Cl₂ and washed withsaturated Na₂CO₃. The aqueous layer was extracted with CH₂Cl₂ fourtimes. The combined organic layers were dried (MgSO₄), concentrated andpurified by flash chromatography to provide the desired product (304.4mg, 40%). MS calculated for C₂₄H₃₆F₃N₄O₄: (M+H) 501.3; found 501.3.

Step E

(1R,3S)-3-(Ethoxymethyl)-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentanamineDihydrochloride

tert-Butyl[(3S)-3-(ethoxymethyl)-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate(295 mg, 0.589 mmol) was dissolved in a 2 M solution of hydrogenchloride in ether (10 mL). After being stirred at room temperatureovernight, the reaction mixture was concentrated under vacuum to providethe desired product (330 mg) as a light yellow solid. MS calculated forC₁₉H₂₈F₃N₄O₂: (M+H) 401.3; found 401.3.

Step F

(4R)-N-[(1R,3S)-3-(Ethoxymethyl)-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amineBis(trifluoroacetate)

To a solution of(1R,3S)-3-(ethoxymethyl)-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentanaminedihydrochloride (100.0 mg, 0.211 mmol),3-methoxytetrahydro-4H-pyran-4-one (82.5 mg, 0.634 mmol) andtriethylamine (0.118 mL, 0.845 mmol) in dry methylene chloride (8.6 mL)was added sodium triacetoxyborohydride (134.3 mg, 0.634 mmol). Afterbeing stirred at room temperature under N₂ overnight, the reaction wasquenched with aqueous NaHCO₃ and diluted with methylene chloride. Theorganic layer was separated and the aqueous layer was extracted withmethylene chloride three times. The organics were combined, dried overMgSO₄, filtered, purified by silica gel Combi-Flash system (gradient, 0to 40% MeOH in EtOAc, 12 gram column) and converted to TFA salt to givethe desired product (115.4 mg, 74%). MS calculated for C₂₅H₃₇F₃N₄O₄:(M+H) 515.3; found 515.4.

Example 30 Preparation of(4R)-N-[(1R,3S)-3-(ethoxymethyl)-3-(4-[4-(trifluoromethyl)pyrimidin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-aminebis(trifluoroacetate)

The title compound was prepared using procedures analogous to thosedescribed for Example 29. MS calculated for C₂₄H₃₆F₃N₅O₄: (M+H) 516.3;found 516.4.

Example 31 Preparation of(4R)-3-methoxy-N-[(1R,3S)-3-(methoxymethyl)-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]tetrahydro-2H-pyran-4-aminebis(trifluoroacetate

The title compound was prepared using procedures analogous to thosedescribed for Example 29. MS calculated for C₂₄H₃₅F₃N₄O₄: (M+H) 501.3;found 501.3.

Example 32 Preparation of(4R)-3-methoxy-N-[(1R,3S)-3-(methoxymethyl)-3-(4-[4-(trifluoromethyl)pyrimidin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]tetrahydro-2H-pyran-4-aminebis(trifluoroacetate)

The title compound was prepared using procedures analogous to thosedescribed for Example 29, MS calculated for C₂₃H₃₄F₃N₅O₄: (M+H) 502.3;found 502.3.

Example 33 Preparation of(4R)-3-methoxy-N-[(1R,3S)-3-[(3R)-tetrahydrofuran-3-yl]-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]tetrahydro-2H-pyran-4-amine

Step A

(3R)-3-Iodotetrahydrofuran

To a solution of (S)-(+)-3-hydroxytetrahydrofuran (0.50 g, 5.7 mmol) inmethylene chloride (50 mL) was added triphenylphosphine (3.0 g, 11mmol), 1H-imidazole (0.75 g, 11 mmol), and iodine (2.9 g, 11 mmol)sequentially. After being refluxed under N₂ overnight, the reaction wasquenched with 0.2 M Na₂S₂O₃ (60 mL). The organic layer was separated andthe aqueous layer was extracted with methylene chloride three times. Thecombined organics were dried (MgSO₄), filtered, and concentrated to givea wet, yellow solid. To the solids was added pentane (100 mL) andstirred for 2 hours. The solids were filtered off and the filtrate wasconcentrated to give the desired product (970 mg, 79.4%) as a yellowoil. ¹H NMR (CDCl₃) δ 4.30-3.85 (5H, m), 2.50-2.20 (2H, m).

Step B

Methyl(1R,4S)-4-[(tert-Butoxycarbonyl)amino]-1-[(3R)-tetrahydrofuran-3-yl]cyclopent-2-ene-1-carboxylate

To a 1.00 M solution of lithium hexamethyldisilazide in tetrahydrofuran(34.8 mL, 34.8 mmol) under N₂ at −78° C. was added a solution of methyl(1R,4S)-4-[(tert-butoxycarbonyl)amino]cyclopent-2-ene-1-carboxylate (4.0g, 16 mmol) in tetrahydrofuran (20 mL). The resulting brown solution wasstirred at −78° C. for 30 min before adding a solution of(3R)-3-iodotetrahydrofuran (3.75 g, 17.4 mmol) in THF (3 mL). Themixture was stirred for 10 min at −78° C. then kept in a freezer readingat −25° C. overnight. The reaction was quenched with saturated ammoniumchloride, extracted with ether three times. The combined extracts weredried (MgSO₄), filtered, concentrated and purified by flash columnchromatography (EtOAc/Hexane) to provide the desired product (1.6 g,31%). MS calculated for C₁₆H₂₅NO₅: (M+H) 312.2; found 312.2.

Step C

(1R,4S)-4-[(tert-Butoxycarbonyl)amino]-1-[(3R)-tetrahydrofuran-3-yl]cyclopent-2-ene-carboxylicAcid

To a solution of methyl(1R,4S)-4-[(tert-butoxycarbonyl)amino]-1-[(3R)-tetrahydrofuran-3-yl]cyclopent-2-ene-1-carboxylate(1.60 g, 5.14 mmol) in tetrahydrofuran (27.8 mL), methanol (27.8 mL) andwater (6.0 mL) was added lithium hydroxide, monohydrate (0.346 g, 8.25mmol). The pink mixture was heated to reflux for 18 h. The organicsolvents were removed in vacuo and the aqueous layer was extracted oncewith ether and then acidified slowly with 6 M HCl until the pH reached3-4. The resulting suspension was extracted with CH₂Cl₂ three times. Thecombined organic layers were dried over MgSO₄, filtered and concentratedto give the product as a mixture of two cis/trans isomers (1.59 g) as alight yellow solid. MS calculated for C₁₅H₂₄NO₅: (M+H) 298.2; found198.2 (M+H-Boc).

Step D

(1S,3R)-3-[(tert-Butoxycarbonyl)amino]-1-[(3R)-tetrahydrofuran-3-yl]cyclopentanecarboxylicAcid

(1R,4S)-4-[(tert-Butoxycarbonyl)amino]-1-[(3R)-tetrahydrofuran-3-yl]cyclopent-2-ene-1-carboxylicacid (0.79 g, 2.6 mol) was dissolved in ethanol (20.0 mL),degassed-purged with N₂, followed by the addition of platinum dioxide(0.150 g, 0.528 mol). The reaction mixture was placed on a Parrapparatus and hydrogenated under hydrogen at 55 psi for 18 h. Themixture was filtered through celite pad, washed with MeOH, concentratedto provide the desired product (730 mg, 91.8%). MS calculated forC₁₅H₂₆NO₅ (M+H) 300.2; found 200.2 (M+H-Boc).

Step E

tert-Butyl[(1R,3S)-3-[(3R)-Tetrahydrofuran-3-yl]-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate

(1S,3R)-3-[(tert-Butoxycarbonyl)amino]-1-[(3R)-tetrahydrofuran-3-yl]cyclopentanecarboxylicacid (350.0 mg, 1.169 mmol),1-[4-(trifluoromethyl)pyridin-2-yl]piperazine dihydrochloride (391.1 mg,1.286 mmol), triethylamine (0.652 mL, 4.68 mmol) andO-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(665.1 mg, 1.754 mol) (HBTU) were mixed in dry DMF (8.0 mL). After beingstirred at room temperature overnight, the reaction mixture was dilutedwith CH₂Cl₂ and washed with saturated Na₂CO₃. The aqueous layer wasextracted with CH₂Cl₂ four times. The combined organic layers were dried(MgSO₄), concentrated and purified by flash chromatography (Combi-flashsystem, 0-50% EtOAc in hexanes, gradient elution, 40 gram column) toprovide the desired product (270 mg, 45%). MS calculated forC₂₅H₃₆F₃N₄O₄ (M+H) 513.3; found 513.3.

Step F

(1R,3S)-3-[(3R)-Tetrahydrofuran-3-yl]-3-(4-[4-(trifluoromethylpyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentanamineDihydrochloride

tert-Butyl[(1R,3S)-3-[(3R)-tetrahydrofuran-3-yl]-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]carbamate(260 mg, 0.51 mmol) was dissolved in a 2 M solution of hydrogen chloridein ether (8 mL). After being stirred at room temperature overnight, thereaction mixture was concentrated under vacuum to provide the desiredproduct (290 mg) as a light yellow solid. MS calculated forC₂₀H₂₇F₃N₄O₂: (M+H) 413.2; found 413.0.

Step G

(4R)-3-Methoxy-N-[(1R,3S)-3-[(3R)-tetrahydrofuran-3-yl]-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]tetrahydro-2H-pyran-4-amine

To a solution of(1R,3S)-3-[(3R)-tetrahydrofuran-3-yl]-3-(4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-ylcarbonyl)cyclopentanaminedihydrochloride (73.8 mg, 0.152 mmol),3-methoxytetrahydro-4H-pyran-4-one (59.4 mg, 0.456 mmol) andtriethylamine (0.0848 mL, 0.608 mmol) in dry methylene chloride (4.1 mL)was added sodium triacetoxyborohydride (96.7 mg, 0.456 mmol). Afterbeing stirred at room temperature under N₂, the reaction was quenchedwith aqueous NaHCO₃ and diluted with CH₂C₂. The organic layer wasseparated and the aqueous layer was extracted with CH₂Cl₂ three times.The organics were combined; dried over MgSO₄, filtered, concentrated andpurified by silica gel chromatography (Combi-Flash system, 0 to 40% MeOHin EtOAc, gradient, 12 gram column) to provide the desired product (20mg, 41%). MS calculated for C₂₆H₃₇F₃N₄O₄: (M+H) 527.3; found 527.3.

Example 34 Preparation of(4R)-3-methoxy-N-[(1R,3S)-3-[(3R)-tetrahydrofuran-3-yl]-3-(4-[4-(trifluoromethyl)pyrimidin-2-yl]piperazin-1-ylcarbonyl)cyclopentyl]tetrahydro-2-pyran-4-amine

The title compound was prepared using procedures analogous to thosedescribed for Example 33. MS calculated for C₂₅H₃₆F₃N₅O₄: (M+H) 528.3;found 528.3.

Example A CCR2 In Vitro Assays

The capacity of the novel compounds of the invention to antagonizechemokine receptor (e.g., CCR2) function can be determined using asuitable screen (e.g., high through-put assay). For example, an agentcan be tested in an extracellular acidification assay, calcium fluxassay, ligand binding assay or chemotaxis assay (see, for example,Hesselgesser et al., J Biol. Chem. 273(25):15687-15692 (1998); WO00/05265 and WO 98/02151).

In a suitable assay, a CCR2 protein which can be isolated orrecombinantly derived is used which has at least one property, activityor functional characteristic of a mammalian CCR2 protein. The specificproperty can be a binding property (to, for example, a ligand orinhibitor), a signalling activity (e.g., activation of a mammalian Gprotein, induction of rapid and transient increase in the concentrationof cytosolic free calcium [Ca⁺⁺]i, cellular response function (e.g.,stimulation of chemotaxis or inflammatory mediator release byleukocytes), and the like.

In an example binding assay, a composition containing a CCR2 protein orvariant thereof is maintained under conditions suitable for binding. TheCCR2 receptor is contacted with a compound to be tested, and binding isdetected or measured.

In an example cell-based assay, cells are used which are stably ortransiently transfected with a vector or expression cassette having anucleic acid sequence which encodes the CCR2 receptor. The cells aremaintained under conditions appropriate for expression of the receptorand are contacted with an agent under conditions appropriate for bindingto occur. Binding can be detected using standard techniques. Forexample, the extent of binding can be determined relative to a suitablecontrol. Also, a cellular fraction, such as a membrane fraction,containing the receptor can be used in lieu of whole cells.

Detection of binding or complex formation in an assay can be detecteddirectly or indirectly. For example, the agent can be labeled with asuitable label (e.g., fluorescent label, label, isotope label, enzymelabel, and the like) and binding can be determined by detection of thelabel. Specific and/or competitive binding can be assessed bycompetition or displacement studies, using unlabeled agent or a ligandas a competitor.

The CCR2 antagonist activity of compounds of the invention can bereported as the inhibitor concentration required for 50% inhibition(IC₅₀ values) of specific binding in receptor binding assays using¹²⁵I-labeled MCP-1, as ligand, and Peripheral Blood Mononuclear Cells(PBMCs) prepared from normal human whole blood via density gradientcentrifugation. Specific binding is preferably defined as the totalbinding (e.g., total cpm on filters) minus the non-specific binding.Non-specific binding is defined as the amount of cpm still detected inthe presence of excess unlabeled competitor (e.g., MCP-1).

Example B Binding Assay

Human PBMCs were used to test compounds of the invention in a bindingassay. For example, 200,000 to 500,000 cells were incubated with 0.1 to0.2 nM ¹²⁵I-labeled MCP-1, with or without unlabeled competitor (10 nMMCP-1) or various concentrations of compounds to be tested. ¹²⁵I-labeledMCP-1, were prepared by suitable methods or purchased from commercialvendors (Perkin Elmer, Boston Mass.). The binding reactions wereperformed in 50 to 250 μL of a binding buffer consisting of 1M HEPES pH7.2, and 0.1% BSA (bovine serum albumin), for 30 min at roomtemperature. The binding reactions were terminated by harvesting themembranes by rapid filtration through glass fiber filters (Perkin Elmer)which was presoaked in 0.3% polyethyleneimine or Phosphate BufferedSaline (PBS). The filters were rinsed with approximately 600 μL ofbinding buffer containing 0.5 M NaCl or PBS, then dried, and the amountof bound radioactivity was determined by counting on a Gamma Counter(Perkin Elmer).

According to this binding assay protocol, the compounds of the presentinvention have IC₅₀ values less than about 3000 nM.

Example C Chemotaxis Assay

The capacity of compounds of the invention to antagonize CCR2 functionwas determined in a leukocyte chemotaxis assay using human peripheralblood mononuclear cells, in a modified Boyden Chamber (Neuro Probe).500,000 cells in serum free DMEM media (In Vitrogen) were incubated withor without the inhibitors and warmed to 37° C. The chemotaxis chamber(Neuro Probe) was also prewarmed. 400 μL of warmed 10 nM MCP-1 was addedto the bottom chamber in all wells except the negative control which hadDMEM added. An 8 micron membrane filter (Neuro Probe) was placed on topand the chamber lid was closed. Cells were then added to the holes inthe chamber lid which were associated with the chamber wells below thefilter membrane. The whole chamber was incubated at 37° C., 5% CO₂ for30 minutes. The cells were then aspirated off, the chanber lid opened,and the filter gently removed. The top of the filter was washed 3 timeswith PBS and the bottom was left untouched. The filter was air dried andstained with Wright Geimsa stain (Sigma). Filters were counted bymicroscopy. The negative control wells served as background and weresubtracted from all values. Antagonist potency was determined bycomparing the number of cell that migrated to the bottom chamber inwells which contained antagonist, to the number of cells which migratedto the bottom chamber in MCP-1 control wells.

According to this chemotaxis assay, the compounds of the invention haveIC₅₀ values less than about 3000 nM.

Example D CCR5 Expression

A leukophoresis (Biological Specialty, Colmar, Pa.) was obtained fromnormal, drug free donors and peripheral blood mononuclear cells (PBMCs)were isolated via density gradient centrifugation. Monocytes werefurther isolated via centrifugal elutriation. After being washed, themonocytes were re-suspended at 10⁶ cells/ml with RPMI (Invitrogen,Carlsbad, Calif.) supplemented with 10% FBS (Hyclone, Logan, Utah) and10-20 ng/mL of recombinant human IL-10 (R&D systems, Minneapolis, Minn.)and incubated in the same medium at 37° C. with 5% CO₂ for 24-48 hr.CCR5 expression on the IL-10-treated monocytes was then verified bystaining the cells with a PE-conjugated anti-human CCR5 antibody((PharMingen, San Diego, Calif.), followed by FACS analysis usingFACSCalibur (BD Biosciences, Bedford, Mass.).

Example E CCR5 Binding Assay

In a 96 well MultiScreen™ filter plate (Millipore Systems, Billerica,Mass.), 3×10⁵ IL-10-treated monocytes in 150 μL RPMI (Invitrogen,Carlsbad, Calif.) with 20 mM HEPES (Invitrogen, Carlsbad, Calif.) and0.3% BSA (Sigma, St Louis, Mo.) were incubated at room temperature for 1hr. with 0.2 nM ¹²⁵I-MIP-1β (Perkin Elmer, Boston, Mass.) and a seriesconcentrations of compound of the invention. Non-specific binding wasdetermined by incubating the cells with 0.3 μM MIP-1β (R&D Systems,Minneapolis, Minn.). The binding reaction was terminated by harvestingthe cells onto the filter in the plate on a vacuum manifold (MilliporeSystems, Billerica, Mass.). The filter was then washed 5 times with RPMI(Invitrogen, Carlsbad, Calif.) supplemented with 20 mM HEPES(Invitrogen, Carlsbad, Calif.), 0.3% BSA (Sigma, St Louis, Mo.) and 0.4M NaCl on the vacuum manifold, air dried, and peeled from the plate. Thefilter dishes corresponding to the sample wells in a filter plate werepunched out using the Millipore Punch System (Millipore Systems,Billerica, Mass.). The amount of bound radioactivity on each filter dishwas determined by counting on a gamma counter. Specific binding wasdefined as the total binding minus the non-specific binding. The bindingdata were evaluated with Prism (GraphPad Software, San Diego, Calif.).Compounds of the invention were found to have a binding affinity ofabout 1 μM or less according to this assay.

Example F HIV-1 Entry Assay

Replication defective HIV-1 reporter virions are generated bycotransfection of a plasmid encoding the NL4-3 strain of HIV-1 (whichhas been modified by mutation of the envelope gene and introduction of aluciferase reporter plasmid) along with a plasmid encoding one ofseveral HIV-1 envelope genes as described by, for example, Connor et al,Virology, 206 (1995), 935-944. Following transfection of the twoplasmids by calcium phosphate precipitation, the viral supernatants areharvested on day 3 and a functional viral titer determined. These stocksare then used to infect U87 cells stably expressing CD4 and thechemokine receptor CCR5 which have been preincubated with or withouttest compound. Infections are carried out for 2 hours at 37° C., thecells washed and media replaced with fresh media containing compound.The cells are incubated for 3 days, lysed and luciferase activitydetermined. Results are reported as the concentration of compoundrequired to inhibit 50% of the luciferase activity in the controlcultures.

Example G HIV-1 Replication Assay in MT-4 Cells

Inhibition of HIV-1 NL4.3 (or III_(B)) replication assays can be carriedout as previously described (Bridger, et al., J. Med. Chem. 42:3971-3981(1999); De Clercq, et al., Proc. Natl. Acad. Sci. 89:5286-5290 (1992);De Clercq, et al., Antimicrob. Agents Chemother. 38:668-674 (1994);Bridger, et al. J. Med. Chem. 38:366-378 (1995)). To summarize, anti-HIVactivity and cytotoxicity measurements are carried out in parallel andare based on the viability of MT-4 cells that are infected with HIV inthe presence of various concentrations of the test compounds. After theMT-4 cells are allowed to proliferate for 5 days, the number of viablecells are quantified by a tetrazolium-based calorimetric3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MIT)procedure in 96-well microtrays. Results can be quanitited to yield EC₅₀values which represent the concentration required to protect 50% of thevirus-infected cells against viral cytopathicity.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference, including patents, patentapplications, and publications, cited in the present application isincorporated herein by reference in its entirety.

What is claimed is:
 1. A method of modulating activity of a chemokinereceptor comprising contacting said chemokine receptor with a compoundof Formula I:

or pharmaceutically acceptable salt or prodrug thereof, wherein: adashed line indicates an optional bond; W is:

V is N, NO or CR⁵; X is N, NO or CR²; Y is N, NO or CR³; Z is N, NO, orCR⁴; wherein no more than one of V, X, Y and Z is NO; R^(A), R^(A1),R^(B) and R^(B1) are each, independently, H, OH, halo, C₁₋₆ alkyl, C₁₋₆alkenyl, C₁₋₆ alkynyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,heterocyclyl, carbocyclyl, NR¹⁰R₁₂, NR¹⁰CO₂R¹¹; NR¹⁰CONR¹⁰R¹²,NR¹⁰SO₂NR¹⁰R¹², NR¹⁰—SO₂—R¹¹, CN, CONR¹⁰R¹², CO₂R¹⁰, NO₂, SR¹⁰, SOR¹⁰,SO₂R¹⁰, or SO₂—NR¹⁰R¹²; R¹ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆hydroxyalkyl, —(C₀₋₆ alkyl)-O—(C₁₋₆ alkyl), —(C₀₋₆ alkyl)-S—(C₁₋₆alkyl), —(C₀₋₆ alkyl)-(C₃₋₇ cycloalkyl)-(C₀₋₆ alkyl), OH, OR¹⁰, SR¹⁰,COR¹¹, CO₂R¹⁰, CONR¹⁰R¹², CR¹⁰R¹¹CO₂R¹⁰ or CR¹⁰R¹¹OCOR¹⁰; R², R³, R⁴, R⁵and R⁶ are each, independently H, OH, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ thioalkoxy, NR¹⁰R¹², NR¹⁰CO₂R¹¹;NR¹⁰CONR¹⁰R¹², NR¹⁰SO₂NR¹⁰R¹², NR¹⁰—SO₂—R¹¹, heterocyclyl, carbocyclyl,carbocyclyloxy, heterocyclyloxy, CN, NO₂, COR¹¹, CONR¹⁰R¹², CO₂R¹⁰, NO₂,SR¹⁰, SOR¹⁰, SO₂R¹⁰; or SO₂—NR¹⁰R¹²; R⁷ is H or C₁₋₆ alkyl optionallysubstituted by 1-3 substituents selected from halo, OH, CO₂H, CO₂—(C₁₋₆alkyl), or C₁₋₃ alkoxy; R⁸ is C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₆cycloalkyloxy or OH; R^(8′) is H; R⁹ and R^(9′) are each, independently,H, C₁₋₆ alkyl, halo, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₆ cycloalkyl, C₃₋₆cycloalkyloxy, OH, CO₂R¹⁰, OCOR¹⁰, wherein said C₁₋₆ alkyl is optionallysubstituted with one or more substituents selected from F, C₁₋₃ alkoxy,OH or CO₂R¹⁰; or R⁹ and R^(9′) together with the carbon atom to whichthey are attached form a 3-7 membered spirocyclyl group; R¹⁰ is H, C₁₋₆alkyl, benzyl, phenyl, or C₃₋₆ cycloalkyl, wherein said C₁₋₆ alkyl,benzyl, phenyl, or C₃₋₆ cycloalkyl is optionally substituted with 1-3selected from halo, OH, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃haloalkoxy, CO₂H, and CO₂—(C₁₋₆ alkyl); R¹¹ is H, OH, C₁₋₆ alkyl, C₁₋₆alkoxy, benzyl, phenyl, benzyloxy, phenyloxy, C₃₋₆ cycloalkyl or C₃₋₆cycloalkyloxy, wherein said C₁₋₆ alkyl, C₁₋₆ alkoxy, benzyl, phenyl,benzyloxy, phenyloxy, C₃₋₆ cycloalkyl or C₃₋₆ cycloalkyloxy, isoptionally substituted with 1-3 substituents selected from halo, OH,C₁₋₃ alkyl, C₁₋₃ alkoxy, CO₂H, CO₂-(C₁₋₆ alkyl) and CF₃; R¹² is H, C₁₋₆alkyl, benzyl, phenyl, or C₃₋₆ cycloalkyl, wherein said C₁₋₆ alkyl,benzyl, phenyl, or C₃₋₆ cycloalkyl is optionally substituted with 1-3selected from halo, OH, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃haloalkoxy, CO₂H, and CO₂—(C₁₋₆ alkyl); and p is 0 or
 1. 2. The methodof claim 1 wherein said chemokine receptor is CCR2 or CCR5.
 3. Themethod of claim 1 wherein said modulating is inhibiting.
 4. The methodof claim 1 wherein said compound inhibits both CCR2 and CCR5.
 5. Amethod of treating a disease associated with expression or activity of achemokine receptor in a patient comprising administering to said patienta therapeutically effective amount of a compound of Formula I:

or pharmaceutically acceptable salt or prodrug thereof, wherein: adashed line indicates an optional bond; W is:

V is N, NO or CR⁵; X is N, NO or CR²; Y is N, NO or CR³; Z is N, NO, orCR⁴; wherein no more than one of V, X, Y and Z is NO; R^(A), R^(A1),R^(B) and R^(B1) are each, independently, H, OH, halo, C₁₋₆ alkyl, C₁₋₆alkenyl, C₁₋₆ alkynyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,heterocyclyl, carbocyclyl, NR¹⁰R¹², NR¹⁰CO₂R¹¹; NR¹⁰CONR¹⁰R¹²,NR¹⁰SO₂NR¹⁰R¹², NR¹⁰—SO₂—R¹¹, CN, CONR¹⁰R¹², CO₂R¹⁰, NO₂, SR¹⁰, SOR¹⁰,SO₂R¹⁰, or SO₂—NR¹⁰R¹²; R¹ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆hydroxyalkyl, —(C₀₋₆ alkyl)-O—(C₁₋₆ alkyl), —(C₀₋₆ alkyl)-S—(C₁₋₆alkyl), —(C₀₋₆ alkyl)-(C₃₋₇ cycloalkyl)-(C₀₋₆ alkyl), OH, OR¹⁰, SR¹⁰,COR¹¹, CO₂R¹⁰, CONR¹⁰R¹², CR¹⁰R¹¹CO₂R¹⁰ or CR¹⁰R¹¹OCOR¹⁰; R², R³, R⁴, R⁵and R⁶ are each, independently H, OH, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ thioalkoxy, NR¹⁰R¹², NR¹⁰CO₂R¹¹;NR¹⁰CONR¹⁰R¹², NR¹⁰SO₂NR¹⁰R¹², NR¹⁰—SO₂—R¹¹, heterocyclyl, carbocyclyl,carbocyclyloxy heterocyclyloxy, CN, NO₂, COR¹¹, CONR¹⁰R¹², CO₂R¹⁰, NO₂,SR¹⁰, SOR¹⁰, SO₂R¹⁰; or SO₂—NR¹⁰R¹²; R⁷ is H or C₁₋₆ alkyl optionallysubstituted by 1-3 substituents selected from halo, OH, CO₂H, CO₂—(C₁₋₆alkyl), or C₁₋₃ alkoxy; R⁸ is C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₆cycloalkyloxy or OH; R^(8′) is H; R⁹ and R^(9′) are each, independently,H, C₁₋₆ alkyl, halo, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₆ cycloalkyl, C₃₋₆cycloalkyloxy, OH, CO₂R¹⁰, OCOR¹⁰, wherein said C₁₋₆ alkyl is optionallysubstituted with one or more substituents selected from F, C₁₋₃ alkoxy,OH or CO₂R¹⁰; or R⁹ and R^(9′) together with the carbon atom to whichthey are attached form a 3-7 membered spirocyclyl group; R¹⁰ is H, C₁₋₆alkyl, benzyl, phenyl, or C₃₋₆ cycloalkyl, wherein said C₁₋₆ alkyl,benzyl, phenyl, or C₃₋₆ cycloalkyl is optionally substituted with 1-3selected from halo, OH, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃haloalkoxy, CO₂H, and CO₂—(C₁₋₆ alkyl); R¹¹ is H, OH, C₁₋₆ alkyl, C₁₋₆alkoxy, benzyl, phenyl, benzyloxy, phenyloxy, C₃₋₆ cycloalkyl or C₃₋₆cycloalkyloxy, wherein said C₁₋₆ alkyl, C₁₋₆ alkoxy, benzyl, phenyl,benzyloxy, phenyloxy, C₃₋₆ cycloalkyl or C₃₋₆ cycloalkyloxy, isoptionally substituted with 1-3 substituents selected from halo, OH,C₁₋₃ alkyl, C₁₋₃ alkoxy, CO₂H, CO₂—(C₁₋₆ alkyl) and CF₃; R¹² is H, C₁₋₆alkyl, benzyl, phenyl, or C₃₋₆ cycloalkyl, wherein said C₁₋₆ alkyl,benzyl, phenyl, or C₃₋₆ cycloalkyl is optionally substituted with 1-3selected from halo, OH, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃haloalkoxy, CO₂H, and CO₂—(C₁₋₆ alkyl); and p is 0 or
 1. 6. The methodof claim 5 wherein said disease is an eye disorder.
 7. The method ofclaim 6 wherein said eye disorder is characterized by dryness of theeye.
 8. A method of treating dryness of the eye in a patient comprisingadministering to said patient a therapeutically effective amount of acompound of Formula I:

or pharmaceutically acceptable salt or prodrug thereof, wherein: adashed line indicates an optional bond; W is:

V is N, NO or CR⁵; X is N, NO or CR²; Y is N, NO or CR³; Z is N, NO, orCR⁴; wherein no more than one of V, X, Y and Z is NO; R^(A), R^(A1),R^(B) and R^(B1) are each, independently, H, OH, halo, C₁₋₆ alkyl, C₁₋₆alkenyl, C₁₋₆ alkynyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,heterocyclyl, carbocyclyl, NR¹⁰R¹², NR¹⁰CO₂R¹¹; NR¹⁰CONR¹⁰R¹²,NR¹⁰SO₂NR¹⁰R¹², NR¹⁰—SO₂—R¹¹, CN, CONR¹⁰R¹², CO₂R¹⁰, NO₂, SR¹⁰, SOR¹⁰,SO₂R¹⁰, or SO₂—NR¹⁰R¹²; R¹ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆hydroxyalkyl, —(C₀₋₆ alkyl)-O—(C₁₋₆ alkyl), —(C₀₋₆ alkyl)-S—(C₁₋₆alkyl), —(C₀₋₆ alkyl)-(C₃₋₇ cycloalkyl)-(C₀₋₆ alkyl), OH, OR¹⁰, SR¹⁰,COR¹¹, CO₂R¹⁰, CONR¹⁰R¹², CR¹⁰R¹¹CO₂R¹⁰ or CR¹⁰R¹¹OCOR¹⁰; R², R³, R⁴, R⁵and R⁶ are each, independently H, OH, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ thioalkoxy, NR¹⁰R¹², NR¹⁰CO₂R¹¹;NR¹⁰CONR¹⁰R¹², NR¹⁰SO₂NR¹⁰R¹², NR¹⁰—SO₂—R¹¹, heterocyclyl, carbocyclyl,carbocyclyloxy, heterocyclyloxy, CN, NO₂, COR¹¹, CONR¹⁰R¹², CO₂R¹⁰, NO₂,SR¹⁰, SOR¹⁰, SO₂R¹⁰; or SO₂—NR¹⁰R¹²; R⁷ is H or C₁₋₆ alkyl optionallysubstituted by 1-3 substituents selected from halo, OH, CO₂H, CO₂—(C₁₋₆alkyl), or C₁₋₃ alkoxy; R⁸ is C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₆cycloalkyloxy or OH; R^(8′) is H; R⁹ and R^(9′) are each, independently,H, C₁₋₆ alkyl, halo, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₆ cycloalkyl, C₃₋₆cycloalkyloxy, OH, CO₂R¹⁰, OCOR¹⁰, wherein said C₁₋₆ alkyl is optionallysubstituted with one or more substituents selected from F, C₁₋₃ alkoxy,OH or CO₂R¹⁰; or R⁹ and R^(9′) together with the carbon atom to whichthey are attached form a 3-7 membered spirocyclyl group; R¹⁰ is H, C₁₋₆alkyl, benzyl, phenyl, or C₃₋₆ cycloalkyl, wherein said C₁₋₆ alkyl,benzyl, phenyl, or C₃₋₆ cycloalkyl is optionally substituted with 1-3selected from halo, OH, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃haloalkoxy, CO₂H, and CO₂—(C₁₋₆ alkyl); R¹¹ is H, OH, C₁₋₆ alkyl, C₁₋₆alkoxy, benzyl, phenyl, benzyloxy, phenyloxy, C₃₋₆ cycloalkyl or C₃₋₆cycloalkyloxy, wherein said C₁₋₆ alkyl, C₁₋₆ alkoxy, benzyl, phenyl,benzyloxy, phenyloxy, C₃₋₆ cycloalkyl or C₃₋₆ cycloalkyloxy, isoptionally substituted with 1-3 substituents selected from halo, OH,C₁₋₃ alkyl, C₁₋₃ alkoxy, CO₂H, CO₂—(C₁₋₆ alkyl) and CF₃; R¹² is H, C₁₋₆alkyl, benzyl, phenyl, or C₃₋₆ cycloalkyl, wherein said C₁₋₆ alkyl,benzyl, phenyl, or C₃₋₆ cycloalkyl is optionally substituted with 1-3selected from halo, OH, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃haloalkoxy, CO₂H, and CO₂—(C₁₋₆ alkyl); and p is 0 or 1.